Chimeric vaccines

ABSTRACT

The invention provides chimeric proteins and nucleic acids encoding these which can be used to generate vaccines against selected antigens. In one aspect, a chimeric protein comprises an antigen sequence and a domain for trafficking the protein to an endosomal compartment, irrespective of whether the antigen is derived from a membrane or non-membrane protein. In one preferred aspect, the trafficking domain comprises a lumenal domain of a LAMP polypeptide. Alternatively, or additionally, the chimeric protein comprises a trafficking domain of an endocytic receptor (e.g., such as DEC-205 or gp200-MR6). The vaccines (DNA, RNA or protein) can be used to modulate or enhance an immune response against any kind of antigen. In one preferred aspect, the invention provides a method for treating a patient with cancer by providing a chimeric protein comprising a cancer-specific antigen or a nucleic acid encoding the protein to the patient.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/824,835, filed Jun. 28, 2010, which is a Continuation of U.S.application Ser. No. 10/474,371, filed Mar. 5, 2004, which is the U.S.national phase, pursuant to 35 U.S.C. §371, of PCT internationalapplication Ser. No. PCT/US2002/010757, Filed Apr. 5, 2002, designatingthe United States and published in English on Oct. 17, 2002 aspublication WO 2002/080851, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional Application Nos. 60/281,607, filed Apr. 5,2001, U.S. Provisional Application 60/281,608, filed Apr. 5, 2001 andU.S. Provisional Application 60/281,621, filed Apr. 5, 2001. Theentireties of these applications are incorporated by reference herein.

GOVERNMENT GRANTS

The work contained in this application was performed under governmentgrant A1 41908 from the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to chimeric vaccines comprising antigen sequencesand trafficking domains, nucleic acids encoding the same, and methodsfor using the same.

BACKGROUND OF THE INVENTION

Antigen recognition and response in the mammalian immune system isgoverned, in part, by the interaction between T-cells and antigenpresenting cells. Via its heterodimeric T cell receptor, a T cellrecognizes peptide fragments of antigens presented as a complex withmajor histocompatibility (MHC) molecules (Yewdell and Bennenk, Cell 62:203, 1990; Davis and Bjorkman, Nature 334: 395, 1988). There are twoparallel cellular systems of T cells and antigen presenting moleculeswhich distinguish between two types of antigens, foreign antigensintroduced from outside of the cell (such as foreign chemicals,bacteria, and toxins) and endogenous antigens produced within the cell(such as viruses or oncogene products) (Bevan, Nature 325: 192, 1987;Braciale, et al., Immunol. Rev. 98:95, 1987; Germain, Nature 322: 687,1986).

There are two general classes of MHC molecules, MHC class I, and MHCclass II molecules. MHC class I molecules present peptide antigensgenerally derived from endogenously produced proteins to the CD8⁺ T_(c)cells, the predominant cytotoxic T cell that is antigen specific. TheMHC class I-related proteolytic system is present in virtually all cellsfor the purpose of degrading highly abnormal proteins and short-livedmolecules or viral proteins. This proteolysis is thought to benon-lysosomal and to involve ATP-dependent covalent conjugation to thepolypeptide ubiquitin (Goldberg, et al., Nature 357: 375, 1992). Peptidefragments, possibly in association with a larger proteasome complex, arethen postulated to enter into the endoplasmic reticulum or some othertype of exocytic compartment (other than the endocytic/lysosomalcompartment). There they bind to MHC class I molecules and follow theconstitutive secretory pathway from the endoplasmic reticulum throughthe Golgi to the cell surface where they are presented by the MHC Iprotein to the CD3-CD8 cytotoxic T cell antigen receptor.

MHC class II molecules generally present antigens that are introducedfrom outside the cells in a process that involves cellular uptake ofmolecules comprising the antigens, and generation of antigenic peptidefragments in endosomal/lysosomal organelles. The MHC class II-relatedprocess by which foreign antigens are processed in antigen presentingcells (APC) cells is generally believed to occur in an endocyticpathway. Antigens taken into the cell by fluid-phase pinocytosis,absorptive endocytosis, or phagocytosis enter into a lateendosomal/lysosomal compartment where large molecules are converted topeptides by digestion through proteases and other hydrolases. Duringthis process, the immunodominant smaller peptides come in contact withand are bound by MHC class II molecules and the peptides are carried tothe cell surface. On the cell surface of APC, these short peptides inconjunction with MHC class II molecules bind the CD3-CD4 complex on thesurface of helper T cells, activating the replication and immunefunction of these cells. Following this interaction, helper T cellsrelease lymphokines that stimulate the proliferation and differentiationof leukocytes and inhibit their emigration from the site of infection.In general, the activation of helper T cells by peptide-loaded APC isrequired for optimal B cell and T cell action, and thus is necessary forproper immune system function.

The exact site of antigen processing and association of processedpeptides with MHC class II in the endosomal/lysosomal pathway is as yetunclear. Data have been presented suggesting that MHC class II moleculesmeet with endocytosed proteins in the early endosomal compartment(Guagliardi, et al., Nature 343: 133, 1990). Partially processedantigens and easily degradable antigens may yield peptides that cancombine with MHC class II in the early endosomal compartment. However,evidence is mounting that the major site of antigen processing andassociation with MHC class II occurs either in the late endosome, thelysosome, or a distinct compartment related to the lysosome (Neefjes, etal., Cell 61: 171, 1990).

The functions of the two types of T cells are significantly different,as implied by their names. Cytotoxic T cells eradicate intracellularpathogens and tumors by direct lysis of cells and by secreting cytokinessuch as γ interferon. Helper T cells also can lyse cells, but theirprimary function is to secrete cytokines that promote the activities ofB cells (antibody-producing cells) and other T cells and thus theybroadly enhance the immune response to foreign antigens, includingantibody-mediated and T_(c)-mediated response mechanisms.

CD4⁺ T cells are the major helper T cell phenotype in the immuneresponse. Their predominant function is to generate cytokines whichregulate essentially all other functions of the immune response. Animalsdepleted of CD4⁺ or humans depleted of CD4⁺ cells (as in patients withAIDS) fail to generate antibody responses, cytotoxic T cell responses,or delayed type hypersensitivity responses. It is well known in the artthat helper T cells are critical in regulating immune responses.

CD4⁺ MHC class II restricted cells have also been shown to havecytotoxic capacity in a number of systems. One of the most importantdisease-relevant cases in which CD4⁺ cytotoxic T cells have beendemonstrated is in the response to fragments of the HIV gp120 protein(Polydefkis, et al., J. Exp. Med. 171: 875, 1990). CD4⁺ MHC class IIrestricted cells also have been shown to be critical in generatingsystemic immune responses against tumors. In an adoptive transfer model,CD4⁺ cells are critical in eliminating FBL tumors in mice. In the activeimmunotherapy model of Golumbek, et al. Science 254: 713, 1991, CD4⁺cells have also been shown to be critical in the systemic immuneresponse against a number of different solid malignancies.

Because CD4⁺ MHC class II restricted cells appear to be the criticalmemory cells in the T cell arm of the immune response, an appropriatevaccination strategy is to generate CD4⁺ antigen-specific MHC classII-restricted memory T cell populations.

Traditional vaccines rely on whole organisms, either pathogenic strainsthat have been killed or strains with attenuated pathogenicity. On theone hand, these vaccines run the risk of introducing the disease theyare designed to prevent if the attenuation is insufficient or if enoughorganisms survive the killing step during vaccine preparation. On theother hand, such vaccines have reduced infectivity and are ofteninsufficiently immunogenic, resulting in inadequate protection from thevaccination.

Recently, molecular biological techniques have been used in an attemptto develop new vaccines based on individual antigenic proteins from thepathogenic organisms. Conceptually, use of antigenic peptides ratherthan whole organisms would avoid pathogenicity while providing a vaccinecontaining the most immunogenic epitopes. However, it has been foundthat pure peptides or carbohydrates tend to be weak immunogens, seemingto require a chemical adjuvant in order to be properly processed andefficiently presented to the immune system. A vaccine dependent on Tcell responses should contain as many T cell epitopes as would be neededto stimulate immunity in a target population of diverse MHC types.Further, since T cell recognition requires intracellular proteinprocessing, vaccine preparations facilitating internalization andprocessing of antigen should generate a more effective immune response.Previous attempts to direct antigens to MHC molecules (see, U.S. Pat.No. 4,400,276) were not effective because the antigen-processing stepwas evaded. A successful hepatitis B vaccine has been prepared usingcloned surface antigen of the hepatitis B virus, but this appears to bedue to the tendency of the hepatitis surface antigen molecule toaggregate, forming regular particles that are highly immunogenic.

Genetic (DNA) vaccines are new and promising candidates for thedevelopment of both prophylactic and therapeutic vaccines. They areproven to be safe and the lack of immune responses to a vector backbonemay be a definitive advantage if repetitive cycles of vaccination arerequired to achieve clinical benefits. However, one potentialdisadvantage of conventional DNA vaccines is their low immunogenicity inhumans. One likely cause of this low immunogenicity is the restrictedaccess of antigens formed within cells to the MHC II pathway for antigenprocessing and presentation to T helper cells.

U.S. Pat. No. 5,633,234 describe chimeric proteins comprising anantigenic domain and a cytoplasmic endosomal/lysosomal targeting signalwhich effectively target antigens to that compartment. The antigenicdomain was processed and peptides from it presented on the cell surfacein association with major histocompatibility (MHC) class II molecules.The cytoplasmic tail of LAMP-1 were used to form the endosomal/lysosomaltargeting domain of the chimeric protein.

SUMMARY OF THE INVENTION

It is an object of this invention to provide vaccines with enhancedimmunogenicity, particularly, genetic vaccines such as DNA or RNAvaccines.

It is a further object of this invention to provide more effectivemethods of vaccination, through the use of immunogens (regardless ofwhether they are derived from membrane or non-membrane proteins) whichare directed to the lysosomal/endosomal compartment and relatedorganelles (e.g., such as MIIC, CIIV, melanosomes, secretory granules,Birbeck granules, and the like) where they are processed and presentedto major histocompatibility complex (MHC) class II molecules so thathelper T cells are preferentially stimulated.

It is yet another object of this invention to provide improved methodsof treatment for cancer by eliciting an anti-tumor immune responsethrough stimulation of helper T cells.

In one aspect, the invention provides a chimeric protein, comprising: anN-terminal domain comprising at least one epitope of an antigen; and atrafficking domain; wherein the trafficking domain directs both membraneand non-membrane proteins to an endosomal compartment (e.g., a lysosome)in a cell. Preferably, the trafficking domain comprises the lumenaldomain of a LAMP polypeptide, such as a LAMP-1 or LAMP-2 polypeptide.

In another aspect, the chimeric protein comprises a targeting sequencethat directs the protein to an endosomal/lysosomal compartment or arelated organelle for protein processing and peptide epitope binding toMHC II, such as the tetrapeptide sequence Tyr-Xaa-Xaa-Xbb, wherein Xaais any amino acid and Xbb is a hydrophobic amino acid. In still anotheraspect, the targeting sequence comprises a dileucine sequence. In afurther aspect, the targeting sequence comprises a cytosolic proteintargeting domain from an endocytic receptor. Suitable domains, include,but are not limited to the targeting domain of a C-type lectin receptor,a DEC-205 polypeptide, gp200-MR6 protein, or homolog, ortholog, variant,or modified form thereof. MR6 In one preferred aspect, the chimericprotein further comprises a Gag polypeptide. In one aspect, the Gagpolypeptide is inserted into a portion of the lumenal domain of a LAMPpolypeptide. More preferably, the protein further comprises atransmembrane domain and/or a signaling domain.

In one particularly preferred aspect, the protein comprises the lumenaldomain of a LAMP polypeptide and a cytoplasmic domain comprising thetetrapeptide sequence Tyr-Xaa-Xaa-Xbb, wherein Xaa is any amino acid andXbb is a hydrophobic amino acid. The chimeric protein, preferably,further comprises a transmembrane protein. Still more preferably, thechimeric protein also comprises a signaling domain.

Any type of antigen may be used to generate chimeric proteins. In oneaspect, the antigen is selected from the group consisting of: a portionof an antigenic material from a pathogenic organism, a portion of anantigenic material from a cancer-specific polypeptide, and a portion ofan antigenic material from a molecule associated with an abnormalphysiological response (e.g., such as an autoimmune disease, an allergicreaction, cancer, or a congenital disease) or a response to a transplantor graft procedure. In another aspect, the antigenic material is from apathogenic organism which is a virus, microorganism, or parasite. In afurther aspect, the virus is an HIV virus. More than one antigen may beincluded in any chimeric protein.

The invention further provides a nucleic acid molecule encoding any ofthe chimeric proteins described above. The invention also provides avector comprising the nucleic acid wherein the nucleic acid molecule isoperably linked to an expression control sequence. In one preferredaspect, the vector is a vaccine vector, suitable for vaccinating apatient against the antigen. In another aspect, the invention provides adelivery vehicle comprising the nucleic acid molecule for facilitatingthe introduction of the nucleic acid molecule into a cell. The deliveryvehicle may be lipid-based (e.g., a liposome formulation), viral-based(e.g., comprising viral proteins encapsulating the nucleic acidmolecule), or cell-based. In one preferred aspect, the vector is avaccine vector.

The invention also provides a cell comprising any of the vectorsdescribed above. In one aspect, the cell is an antigen presenting cell.The antigen presenting cell may be a professional antigen presentingcell (e.g., a dendritic cell, macrophage, B cell, and the like) or anengineered antigen presenting cell (e.g., a non-professional antigenpresenting cell engineered to express molecules required for antigenpresentation, such as MHC class II molecules). The molecules requiredfor antigen presentation may be derived from other cells, e.g.,naturally occurring, or may themselves be engineered (e.g., mutated ormodified to express desired properties, such as higher or lower affinityfor an antigenic epitope). In one aspect, the antigen presenting celldoes not express any co-stimulatory signals and the antigen is anauto-antigen.

The invention additionally provides a kit comprising a plurality ofcells comprising any of the vectors described above. At least two of thecells express different MHC class II molecules, and each cell comprisesthe same vector. In one aspect, a kit is provided comprising a vectorand a cell for receiving the vector.

The invention also provides a transgenic animal comprising at least oneof the cells described above.

The invention further provides a method for generating an immuneresponse in an animal to an antigen, comprising: administering to theanimal a cell as described above, wherein the cell expresses, or can beinduced to express, the chimeric protein in the animal. In one aspect,the cell comprises an MHC class II molecule compatible with MHC proteinsof the animal, such that the animal does not generate an immune responseagainst the MHC class II molecule. In one preferred aspect, the animalis a human.

In one aspect, the invention provides a method for eliciting an immuneresponse to an antigen, comprising administering to an animal, any ofthe vectors described above. Preferably, the vector is infectious for acell of the animal. For example, the vector may be a viral vector, suchas a vaccinia vector. The antigen may be selected from the groupconsisting of: a portion of an antigenic material from a pathogenicorganism, a portion of an antigenic material from a cancer-specificpolypeptide, and a portion of an antigenic material from a moleculeassociated with an abnormal physiological response or a transplantationantigen. In one aspect, the pathogenic organism is a virus,microorganism, or parasite. In another aspect, the virus is an HIVvirus. In still another aspect, the abnormal physiological response isan autoimmune disease, an allergic reaction, cancer, or a congenitaldisease.

In a further aspect, a cell is obtained from a patient, the vector isintroduced into the cell and the cell or progeny of the cell isreintroduced into the patient. In one aspect, the cell is a stem cellcapable of differentiating into an antigen presenting cell. In anotheraspect, the cell does not express any co-stimulatory signals and theantigen is an autoantigen.

BRIEF DESCRIPTION OF THE FIGURES

The objects and features of the invention can be better understood withreference to the following detailed description and accompanyingdrawings.

FIG. 1A is a schematic representation of the different plasmidconstructs. FIG. 1B is a Western blot analysis of COS cells transfectedwith different plasmids probed with anti-Gag). The horizontal arrowsindicate the promoter region of the plasmids and the boxed areasrepresent the open reading frames. The separate boxes represent thedifferent pieces of the protein chimeras indicated in the key below.TM/CYD boxes indicate the transmembrane and cytoplasmic domains includedin the construct. The red diamonds indicate the AAV-ITRs added to theexpression vector.

FIG. 2 shows an anti-HIV lysate antibody response in an average ofindividual mice (n=5). ELISA analysis of antibody binding to protein ofan HIV-1 viral triton-X-100 lysate, 50 ul of 5 ug/ml added to plate.

FIG. 3 shows the titer of anti-HIV lysate antibody response inindividual immunizations.

FIGS. 4A and B show an anti-HIV lysate antibody response at day 29 afterthe first immunization and 15 days after the second immunization;average of individual mice (n=6).

FIG. 5 shows an Interferon gamma and IL4 assay at day 45 afterimmunization with 50 μg DNA on days 0 and 30. Spleen cells werestimulated by medium (control), 5 μg Gag protein. LEFT: p55 Gag proteinspecific IL4 production. RIGHT: p55 Gag-specific INFγ production.

FIG. 6 shows a real-time IL-4 PCR assay at day 45 after immunizationwith 50 μg DNA on days 0 and 30. Spleen cells were stimulated by medium(control), and 5 μg Gag protein.

FIGS. 7A-D show effects of LAMP/Gag chimeras on antigen-specificCD4-mediated cytokine responses. FIG. 7E shows various vectors encodingthe chimeras.

FIG. 8 shows vectors used to monitor trafficking of LAMP/Gag/DECchimeras according to one aspect of the invention.

FIG. 9 shows Western Blot analysis of COS cells transfected withLAMP/Gag/DCLR chimeras.

FIGS. 10A-F show immunofluorescence of cells transfected withLAMP/Gag/DCLR chimeras.

FIGS. 11A-B, C1-C3 show comparisons of immune responses induced byLAMP/Gag/DCLR trafficking.

DETAILED DESCRIPTION

The invention provides chimeric proteins and nucleic acids encodingthese which can be used to generate vaccines against selected antigens.In one aspect, a chimeric protein comprises an antigen sequence and adomain for trafficking the protein to an endosomal/lysosomalcompartmentor related organelle, irrespective of whether the antigen is derivedfrom a membrane or non-membrane protein. In one preferred aspect, thetrafficking domain comprises a lumenal domain of a LAMP polypeptide.Alternatively, or additionally, the chimeric protein comprises atrafficking domain of an endocytic receptor (e.g., such as C-typelectin, DEC-205 or gp200-MR6). The vaccines can be used to modulate orenhance an immune response. In one preferred aspect, the inventionprovides a method for treating a patient with cancer by providing achimeric protein comprising a cancer-specific antigen or a nucleic acidencoding the protein to the patient.

DEFINITIONS

The following definitions are provided for specific terms which are usedin the following written description.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. The term “a nucleic acid molecule” includesa plurality of nucleic acid molecules.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude other elements. “Consisting essentially of”, when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

As used herein, “the lysosomal/endosomal compartment” refers tomembrane-bound acidic vacuoles containing LAMP molecules in themembrane, hydrolytic enzymes that function in antigen processing, andMHC class II molecules for antigen recognition and presentation. Thiscompartment functions as a site for degradation of foreign materialsinternalized from the cell surface by any of a variety of mechanismsincluding endocytosis, phagocytosis and pinocytosis, and ofintracellular material delivered to this compartment by specializedautolytic phenomena (de Duve, Eur. J. Biochem. 137: 391, 1983). The term“endosome” as used herein and in the claims encompasses a lysosome.

As used herein, a “lysosome-related organelle” refers to any organellewhich comprises lysosymes and includes, but is not limited to, MIIC,CIIV, melanosomes, secretory granules, lytic granules, platelet-densegranules, basophil granules, Birbeck granules, phagolysosomes, secretorylysosomes, and the like. Preferably, such an organelle lacks mannose6-phosphate receptors and comprises LAMP, but may or may not comprise anMHC class II molecule. For reviews, see, e.g., Blott and Griffiths,Nature Reviews, Molecular Cell Biology, 2002; Dell'Angelica, et al., TheFASEB Journal 14: 1265-1278, 2000.

As used herein, the terms “polynucleotide” and “nucleic acid molecule”are used interchangeably to refer to polymeric forms of nucleotides ofany length. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example, single-,double-stranded and triple helical molecules, a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA,recombinant polynucleotides, branched polynucleotides, aptamers,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A nucleic acid molecule mayalso comprise modified nucleic acid molecules (e.g., comprising modifiedbases, sugars, and/or internucleotide linkers).

As used herein, the term “peptide” refers to a compound of two or moresubunit amino acids, amino acid analogs, or peptidomimetics. Thesubunits may be linked by peptide bonds or by other bonds (e.g., asesters, ethers, and the like).

As used herein, the term “amino acid” refers to either natural and/orunnatural or synthetic amino acids, including glycine and both D or Loptical isomers, and amino acid analogs and peptidomimetics. A peptideof three or more amino acids is commonly called an oligopeptide if thepeptide chain is short. If the peptide chain is long (e.g., greater thanabout 10 amino acids), the peptide is commonly called a polypeptide or aprotein. While the term “protein” encompasses the term “polypeptide”, a“polypeptide” may be a less than full-length protein.

As used herein a “LAMP polypeptide” refers to LAMP-1, LAMP-2,CD63/LAMP-3, DC-LAMP, or any lysosomal associated membrane protein, orhomologs, orthologs, variants (e.g., allelic variants) and modifiedforms (e.g., comprising one or more mutations, either naturallyoccurring or engineered). In one aspect, a LAMP polypeptide is amammalian lysosomal associated membrane protein, e.g., such as a humanor mouse lysosomal associated membrane protein. More generally, a“lysosomal membrane protein” refers to any protein comprising a domainfound in the membrane of an endosomal/lysosomal compartment orlysosome-related organelle and which further comprises a lumenal domain.

As used herein, “an endocytic receptor” refers to a transmembraneprotein with either its C-terminal or N-terminal facing the cytoplasmand which comprises a trafficking domain (e.g., a lumenal domain) fortransporting a polypeptide or peptide conjugated to it (e.g., via anchemical bond) to an MHC class II molecule or to an intracellularcompartment for subsequent association with an MHC class II molecule.Examples of endocytic receptors include, but are not limited to,Fc-receptors, complement receptors, scavenger receptors, integrins,lectins (e.g., C-type lectins), DEC-205 polypeptides, gp200-MR6polypeptides, Toll-like receptors, heat shock protein receptors (e.g.,CD 91), apoptotic body or necrotic body receptors (e.g., such as CD 14),or homologs, orthologs, variants (e.g., allelic variants) and modifiedforms thereof (e.g., comprising one or more mutations, either naturallyoccurring or engineered).

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or translated intopeptides, polypeptides, or proteins. If the polynucleotide is derivedfrom genomic DNA, expression may include splicing of the mRNAtranscribed from the genomic DNA.

As used herein, “under transcriptional control” or “operably linked”refers to expression (e.g., transcription or translation) of apolynucleotide sequence which is controlled by an appropriatejuxtaposition of an expression control element and a coding sequence. Inone aspect, a DNA sequence is “operatively linked” to an expressioncontrol sequence when the expression control sequence controls andregulates the transcription of that DNA sequence.

As used herein, “coding sequence” is a sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate expression control sequences. The boundaries of a codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, a prokaryotic sequence,cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g.,mammalian) DNA, and even synthetic DNA sequences. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence.

As used herein, two coding sequences “correspond” to each other if thesequences or their complementary sequences encode the same amino acidsequences.

As used herein, “signal sequence” denotes the endoplasmic reticulumtranslocation sequence. This sequence encodes a signal peptide thatcommunicates to a cell to direct a polypeptide to which it is linked(e.g., via a chemical bond) to an endoplasmic reticulum vesicularcompartment, to enter an exocytic/endocytic organelle, to be deliveredeither to a cellular vesicular compartment, the cell surface or tosecrete the polypeptide. This signal sequence is sometimes clipped offby the cell in the maturation of a protein. Signal sequences can befound associated with a variety of proteins native to prokaryotes andeukaryotes.

As used herein, “trafficking” denotes movement or progression of thepolypeptide chimeric antigen through all of the cellular organelles orcompartments in the pathway from the rough endoplasmic reticulum to theendosomal/lysosomal compartment or related organelles where antigenprocessing and binding to MHC II occurs. “Transport” refers to deliveryof a chimeric protein to one particular type of cellular compartment.

As used herein, “targeting” denotes the polypeptide sequence thatdirects the trafficking of the polypeptide chimeric antigen to thepreferred site or cellular organelles or compartment where antigenprocessing and binding to MHC II occurs.

As used herein, “a trafficking domain” refers to a series of continuousor discontinuous amino acids in a protein which are required forvesicular flow of the protein through one or more cellularcompartments/organelles. A trafficking domain preferably comprisesnecessary sequences for proper protein folding to mediate this flow. Inone aspect, a trafficking domain comprises a lumenal sequence;preferably, such a sequence comprises one or more binding sites forinteractions with a cellular folding protein such as a chaperone.

In contrast, as used herein, a “targeting domain” refers to a series ofamino acids which are required for deliver to a cellularcompartment/organelle. Preferably, a targeting domain is a sequencewhich binds to an adaptor or AP protein (e.g., such as an AP1, AP2, orAP3 protein). Exemplary targeting domain sequences are described inDell'Angelica, 2000, supra.

A “chimeric DNA” is an identifiable segment of DNA within a larger DNAmolecule that is not found in association with the larger molecule innature. Thus, when the chimeric DNA encodes a protein segment, thesegment coding sequence will be flanked by DNA that does not flank thecoding sequence in any naturally occurring genome. Allelic variations ornaturally occurring mutational events do not give rise to a chimeric DNAas defined herein.

As used herein, a “nucleic acid delivery vector” is a nucleic acidmolecule which can transport a polynucleotide of interest into a cell.Preferably, such a vector comprises a coding sequence operably linked toan expression control sequence. However, a polynucleotide sequence ofinterest may not necessarily comprise a coding sequence. For example, inone aspect a polynucleotide sequence of interest is an aptamer whichbinds to a target molecule. In another aspect, the sequence of interestis a complementary sequence of a regulatory sequence which binds to aregulatory sequence to inhibit regulation of the regulatory sequence. Instill another aspect, the sequence of interest is itself a regulatorysequence (e.g., for titrating out regulatory factors in a cell).

As used herein, a “nucleic acid delivery vehicle” is defined as anymolecule or group of molecules or macromolecules that can carry insertedpolynucleotides into a host cell (e.g., such as genes or gene fragments,antisense molecules, ribozymes, aptamers, and the like) and which occursin association with a nucleic acid vector as described above.

As used herein, “nucleic acid delivery,” or “nucleic acid transfer,”refers to the introduction of an exogenous polynucleotide (e.g., such asa “transgene”) into a host cell, irrespective of the method used for theintroduction. The introduced polynucleotide may be stably or transientlymaintained in the host cell. Stable maintenance typically requires thatthe introduced polynucleotide either contains an origin of replicationcompatible with the host cell or integrates into a replicon of the hostcell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclearor mitochondrial chromosome.

As used herein, a “viral vector” refers to a virus or viral particlethat comprises a polynucleotide to be delivered into a host cell, eitherin vivo, ex vivo or in vitro. Examples of viral vectors include, but arenot limited to, adenovirus vectors, adeno-associated virus vectors,retroviral vectors, and the like. In aspects where gene transfer ismediated by an adenoviral vector, a vector construct refers to thepolynucleotide comprising the adenovirus genome or part thereof, and aselected, non-adenoviral gene, in association with adenoviral capsidproteins.

As used herein, “adenoviral-mediated gene transfer” or “adenoviraltransduction” refers to the process by which a gene or nucleic acidsequences are transferred into a host cell by virtue of the adenovirusentering the cell. Preferably, the virus is able to replicate and/orintegrate and be transcribed within the cell.

As used herein, “adenovirus particles” are individual adenovirus virionscomprised of an external capsid and internal nucleic acid material,where the capsid is further comprised of adenovirus envelope proteins.The adenovirus envelope proteins may be modified to comprise a fusionpolypeptide which contains a polypeptide ligand covalently attached tothe viral protein, e.g., for targeting the adenoviral particle to aparticular cell and/or tissue type.

As used herein, the term “administering a molecule to a cell” (e.g., anexpression vector, nucleic acid, an accessory factor, a deliveryvehicle, agent, and the like) refers to transducing, transfecting,microinjecting, electroporating, or shooting the cell with the molecule.In some aspects, molecules are introduced into a target cell bycontacting the target cell with a delivery cell (e.g., by cell fusion orby lysing the delivery cell when it is in proximity to the target cell).

As used herein, “hybridization” refers to a reaction in which one ormore polynucleotides react to form a complex that is stabilized viahydrogen bonding between the bases of the nucleotide residues. Thehydrogen bonding may occur by Watson-Crick base pairing, Hoogsteinbinding, or in any other sequence-specific manner. The complex maycomprise two strands forming a duplex structure, three or more strandsforming a multi-stranded complex, a single self-hybridizing strand, orany combination of these. A hybridization reaction may constitute a stepin a more extensive process, such as the initiation of a PCR reaction,or the enzymatic cleavage of a polynucleotide by a ribozyme.

As used herein, a polynucleotide or polynucleotide region (or apolypeptide or polypeptide region) which has a certain percentage (forexample, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99%) of “sequence identity” to another sequencemeans that, when maximally aligned, using software programs routine inthe art, that percentage of bases (or amino acids) are the same incomparing the two sequences.

Two sequences are “substantially homologous” or “substantially similar”when at least about 50%, at least about 60%, at least about 70%, atleast about 75%, andpreferably at least about 80%, and most preferablyat least about 90 or 95% of the nucleotides match over the definedlength of the DNA sequences. Similarly, two polypeptide sequences are“substantially homologous” or “substantially similar” when at leastabout 50%, at least about 60%, at least about 66%, at least about 70%,at least about 75%, and preferably at least about 80%, and mostpreferably at least about 90 or 95% of the amino acid residues of thepolypeptide match over a defined length of the polypeptide sequence.Sequences that are substantially homologous can be identified bycomparing the sequences using standard software available in sequencedata banks. Substantially homologous nucleic acid sequences also can beidentified in a Southern hybridization experiment under, for example,stringent conditions as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.

As used herein, a “trafficking sequence” which is substantiallyhomologous to another trafficking sequence is one which sharessubstantial homology to the other trafficking sequence; however, theultimate test for substantial homology is a functional assay in which apolypeptide comprising a substantially sequence substantially homologousto a trafficking sequence is able to co-localize to the same endosomalcompartment as the trafficking sequence.

“Conservatively modified variants” of domain sequences also can beprovided. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical sequences. Specifically, degenerate codon substitutions can beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res. 19: 5081;Ohtsuka, et al., 1985, J. Biol. Chem. 260: 2605-2608; Rossolini et al.,1994, Mol. Cell. Probes 8: 91-98).

The term “biologically active fragment”, “biologically active form”,“biologically active equivalent” of and “functional derivative” of awild-type protein, possesses a biological activity that is at leastsubstantially equal (e.g., not significantly different from) thebiological activity of the wild type protein as measured using an assaysuitable for detecting the activity. For example, a biologically activefragment comprising a trafficking domain is one which can colocalize tothe same compartment as a full length polypeptide comprising thetrafficking domain.

As used herein, “in vivo” nucleic acid delivery, nucleic acid transfer,nucleic acid therapy” and the like, refer to the introduction of avector comprising an exogenous polynucleotide directly into the body ofan organism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

As used herein, the term “in situ” refers to a type of in vivo nucleicacid delivery in which the nucleic acid is brought into proximity with atarget cell (e.g., the nucleic acid is not administered systemically).For example, in situ delivery methods include, but are not limited to,injecting a nucleic acid directly at a site (e.g., into a tissue, suchas a tumor or heart muscle), contacting the nucleic acid with cell(s) ortissue through an open surgical field, or delivering the nucleic acid toa site using a medical access device such as a catheter.

As used herein, the term “isolated” means separated from constituents,cellular and otherwise, in which the polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, are normallyassociated with in nature. For example, with respect to apolynucleotide, an isolated polynucleotide is one that is separated fromthe 5′ and 3′ sequences with which it is normally associated in thechromosome. As is apparent to those of skill in the art, a non-naturallyoccurring polynucleotide, peptide, polypeptide, protein, antibody, orfragments thereof, does not require “isolation” to distinguish it fromits naturally occurring counterpart.

As used herein, a “target cell” or “recipient cell” refers to anindividual cell or cell which is desired to be, or has been, a recipientof exogenous nucleic acid molecules, polynucleotides and/or proteins.The term is also intended to include progeny of a single cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic or total DNA complement) to the original parent cell due tonatural, accidental, or deliberate mutation. A target cell may be incontact with other cells (e.g., as in a tissue) or may be foundcirculating within the body of an organism.

As used herein, a “subject” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, murines,simians, humans, farm animals, sport animals, and pets.

The terms “cancer,” “neoplasm,” and “tumor,” are used interchangeablyand in either the singular or plural form, refer to cells that haveundergone a malignant transformation that makes them pathological to thehost organism. Primary cancer cells transformation that makes thempathological to the host organism. Primary cancer cells (that is, cellsobtained from near the site of malignant transformation) can be readilydistinguished from non-cancerous cells by well-established techniques,particularly histological examination. The definition of a cancer cell,as used herein, includes not only a primary cancer cell, but any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by procedures such as CAT scan, MR imaging,X-ray, ultrasound or palpation, and/or which is detectable because ofthe expression of one or more cancer-specific antigens in a sampleobtainable from a patient.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin Remington'sPharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)).

A cell has been “transformed”, “transduced”, or “transfected” byexogenous or heterologous nucleic acids when such nucleic acids havebeen introduced inside the cell. Transforming DNA may or may not beintegrated (covalently linked) with chromosomal DNA making up the genomeof the cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element, such as aplasmid. In a eukaryotic cell, a stably transformed cell is one in whichthe transforming DNA has become integrated into a chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations (e.g., at least about 10).

As used herein, an “effective amount” is an amount sufficient to affectbeneficial or desired results, e.g., such as an effective amount ofnucleic acid transfer and/or expression, and/or the attainment of adesired therapeutic endpoint. An effective amount can be administered inone or more administrations, applications or dosages. In one aspect, aneffective amount of a nucleic acid delivery vector is an amountsufficient to transform/transduce/transfect at least one cell in apopulation of cells comprising at least two cells.

As used herein, a “therapeutically effective amount” is used herein tomean an amount sufficient to prevent, correct and/or normalize anabnormal physiological response. In one aspect, a “therapeuticallyeffective amount” is an amount sufficient to reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant feature of pathology, such asfor example, size of a tumor mass, antibody production, cytokineproduction, fever or white cell count, etc.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies (e.g., bispecific antibodies). An“antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen. Exemplary antibody moleculesare intact immunoglobulin molecules, substantially intact immunoglobulinmolecules, and those portions of an immunoglobulin molecule thatcontains the paratope, including Fab, Fab′, F(ab′)₂ and F(v) portions,which portions are preferred for use in the therapeutic methodsdescribed herein.

An “epitope” is a structure, usually made up of a short peptide sequenceor oligosaccharide, that is specifically recognized or specificallybound by a component of the immune system. T-cell epitopes havegenerally been shown to be linear oligopeptides. Two epitopes correspondto each other if they can be specifically bound by the same antibody.Two epitopes correspond to each other if both are capable of binding tothe same B cell receptor or to the same T cell receptor, and binding ofone antibody to its epitope substantially prevents binding by the otherepitope (e.g., less than about 30%, preferably, less than about 20%, andmore preferably, less than about 10%, 5%, 1%, or about 0.1% of the otherepitope binds).

The term “antigenic material” as used herein covers any substance thatwill elicit an innate or adaptive immune response.

The term “antigen presenting cell” as used herein intends any cell whichpresents on its surface an antigen in association with a majorhistocompatibility complex molecule, or portion thereof, or,alternatively, one or more non-classical MHC molecules, or a portionthereof. Examples of suitable APCs are discussed in detail below andinclude, but are not limited to, whole cells such as macrophages,dendritic cells, B cells, hybrid APCs, and foster antigen presentingcells.

As used herein an “engineered antigen-presenting cell” refers to anantigen-presenting cell that has a non-natural molecular moiety on itssurface. For example, such a cell may not naturally have a costimulatoron its surface or may have additional artificial costimulator inaddition to natural costimulator on its surface, or may express anon-natural class II molecule on its surface.

As used herein, “immune effector cells” refers to cells capable ofbinding an antigen and which mediate an immune response. These cellsinclude, but are not limited to, T cells, B cells, monocytes,macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for exampleCTL lines, CTL clones, and CTLs from tumor, inflammatory, or otherinfiltrates.

An “isolated” or “purified” population of cells is substantially free ofcells and materials with which it is associated in nature. Bysubstantially free or substantially purified APCs is meant at least 50%of the population are APCs, preferably at least 70%, more preferably atleast 80%, and even more preferably at least 90% free of non-APCs cellswith which they are associated in nature.

As used herein, a “genetic modification” refers to any addition,deletion or disruption to a cell's normal nucleotides. Any method whichcan achieve the genetic modification of APCs are within the spirit andscope of this invention. Art recognized methods include viral mediatedgene transfer, liposome mediated transfer, transformation, transfectionand transduction, e.g., viral-mediated gene transfer such as the use ofvectors based on DNA viruses such as adenovirus, adeno-associated virusand herpes virus, as well as retroviral based vectors.

The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Maniatis, Fritsch &Sambrook, In Molecular Cloning: A Laboratory Manual (1982); DNA Cloning:A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins, eds., 1985); Transcriptionand Translation (B. D. Hames & S. I. Higgins, eds., 1984); Animal CellCulture (R. I. Freshney, ed., 1986); Immobilized Cells and Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984).

Chimeric Vaccines

In U.S. Pat. No. 5,633,234, antigenic sequences were ligated tocytoplasmic domains of LAMP, transmembrane domains and signal sequencesto form “antigen/LAMP chimeras”. These chimeras were found to bedirected to an endosomal/lysosomal trafficking pathway. With severalmembrane proteins, antigen/LAMP chimeras were found to elicit a muchgreater immune response than wild-type antigen.

This approach has proved useful in increasing cellular and humoralresponses to several virus antigens, human papillomavirus E7, denguevirus membrane protein, HIV-1 gp160 membrane protein, HIV-1 p55 Gag,West Nile membrane protein, hepatitis C virus NS3 protein andcytomegalovirus pp 65 (see, e.g., Bonini, et al., J. Immunol. 166:5250-5257, 2001). The enhanced immune response can be attributed toco-localization of LAMP with MHC II and the more efficient processingand delivery of antigenic peptides. In addition, LAMP-targeting isreported to result in the presentation of an increased number ofimmunogenic epitopes, thus inducing a qualitatively broadened immuneresponse compared to untargeted antigen. For example, Fernandes et al.,2000, Eur. J. Immunol. 30(8): 2333-43, demonstrated an increase in thenumber of presented peptides of a LAMP-trafficked OVA antigen encoded ina vaccinia vector. Of 12 peptides generated from exogenously suppliedOVA, 9 were presented by an OVA/LAMP chimera, as compared to only 2 bythe construct without LAMP.

However, while the cytoplasmic domain of LAMP is necessary (inconjunction with a signal sequence and transmembrane domain), it is notsufficient for endosomal/lysosomal trafficking. It is the discovery ofthe present invention that sequences of the lumenal domain of alysosomal associated membrane protein such as a LAMP polypeptide arealso required for the trafficking of some proteins to the lysosomalvesicular pathway.

For example, viral capsid or non-structural proteins, and other proteinsnot normally are not present in a membrane structure, do not track to avesicular compartment occupied by LAMP and MHC II, and do not elicit anenhanced immune response. It is apparent that trafficking of proteins ina vesicular pathway requires more than a targeting signal signal, likelysequences that are involved in protein folding and interactions withother proteins that are involved in protein vesicular trafficking.

Therefore, the invention provides chimeras which comprise an antigen andlumenal sequences of a polypeptide that result in trafficking of thechimera to the endosomal/lysosomal compartment for antigen processingand antigen epitope association with MHC II. In one aspect, the chimericprotein additionally comprises cytoplasmic targeting sequences thatdirect the chimera to endosomal/lysosomal compartments. Additionally,the chimeric protein also may comprise a signal sequence and/or atransmembrane sequence. Suitable trafficking domains are provide byLAMP-1, LAMP-2, DC-LAMP, Trp-1, DEC-205, gp200-MR6, and otherpolypeptides, as discussed below. The signal sequence and transmembranesequence may, but do not have to be, from these polypeptides. However,in one aspect, an antigen/LAMP chimera comprises a full length LAMPpolypeptide.

Antigen Encoding Sequences

The present invention is widely applicable to antigenic materials thatare of use in vaccines or in other contexts. This antigenic materialwill generally contain peptide segments that can be released bylysosomal enzymes and, when released, correspond to MHC class IIepitopes. The antigenic material may also contain regions that stimulateother components of the immune system, including all immunoglobulinresponses, and MHC I responses.

Because the constructs of the present invention traversepost-translational modification compartments prior to transport to thelysosomal compartment, the antigenic domain may also include epitopesresulting from cellular modification. Essentially, any polypeptide thatcan be synthesized by a mammalian cell and which contains B and T cellepitopes incorporated into antigenic domains, either directly in primaryamino acid sequence or in signals directing its creation duringpost-translational processing may be used as a source of antigenicmaterial.

Selection of the most appropriate portion of the desired antigen proteinfor use as the antigenic domain can be done by functional screening.Broadly, this screening method involves cloning DNA encoding one or moresegments of the protein antigen; and at least a domain for traffickingthe chimera to the endosomal/lysosomal compartment for antigenprocessing and antigen epitope association with MHC II. Preferably, sucha construct will incorporate one or more DNA sequences encoding a signalsequence and/or transmembrane domain and/or a cytoplasmic targetingdomain (e.g., such as the cytoplasmic tail of a LAMP polypeptide). Thecloned DNA is expressed, preferably in an antigen presenting cell line(but not necessarily a professional antigen presenting cell).

The particular screening procedure depends upon the type of antigen andthe assays for its antigenic activity. Antigenicity may be measured bystimulation of antigen-specific MHC class II specific T cell line orclone. Alternatively, antigenicity may be determined by measurement ofthe ability to generate antibodies or T cells specific for the antigenin vivo. These and other tests of antigenic activity are well known tothose skilled in the art.

Antigens that may serve as the source of preferred antigenic materialinclude tumor antigens, auto-antigens, transplantation antigens, cellsurface proteins found on mammalian cells, cancer-specific proteins,proteins associated with abnormal physiological responses, proteins ofbacteria, protozoa or fungi, including especially proteins found in thecell walls or cell membranes of these organisms, and proteins encoded bythe genomes of viruses including retroviruses such as HIV andhepadnaviruses.

Particularly preferred antigens are antigens encoded by the genomes oforganisms causative for, or associated with, hepatitis, rabies, malaria,parasitic infections (e.g., such as schistosomiasis), cancer, AIDS,yellow fever, dengue fever, Japanese encephalitis, West Nile fever,measles, smallpox, anthrax, Ebola, equine encephalitis, Rift valleyfever, cat scratch fever, viral meningitis, plague, tularemia, anddiseases caused by other pathogenic organisms. Particularly preferredviral antigens are virally-encoded proteins encoded by the genome ofviruses pathogenic to man, horses, cows, pigs, llamas, giraffes, dogs,cats or chickens. Non-limiting examples include peptides from theinfluenza nucleoprotein composed of residues 365-80 (NP365-80), NP50-63,and NP147-58 and peptides from influenza hemagglutinin HA202-21 andHA523-45, defined previously in class I restricted cytotoxicity assays(Perkins et al., 1989, J. Exp. Med. 170: 279-289). Peptides representingepitopes displayed by the malarial parasite Plasmodium falciparum havebeen described (see, e.g., U.S. Pat. No. 5,609,872).

Antigenic materials also may include self-antigens recognized intransplant rejection, allergies, hypersensitivity responses, andautoimmune disorders. For example, the acetylcholine receptor (AChR)which is recognized in myasthenia gravis, may provide a source ofantigenic materials. Another class of self-antigens for which antigenicepitopes have been described is human chorionic gonadotropin (hCG) betasubunit (see, e.g., U.S. Pat. No. 5,733,553). These epitopes findutility in contraceptive methods.

Synthetic antigens and altered antigens also can be used in the methodsdescribed herein. Synthetic antigenic peptide epitopes have modifiedamino acid sequences relative to their natural counterparts. Furtherencompassed by the term synthetic antigenic peptide” are multimers(concatemers) of a synthetic antigenic peptides, optionally includingintervening amino acid sequences. For example, synthetic antigenicpeptide epitopes of the present invention can be designed based on knownamino acid sequences of antigenic peptide epitopes.

Other particularly preferred antigens include, but are not limited to,an HIV encoded polypeptide such as Gag, Env, Rev, Tat, and/or Nefpolypeptides, gp160, and the like; papilloma virus core antigen; HCVstructural and non-structural proteins; and CMV structural andnon-structural proteins.

Also included within the scope of the invention are antigenic peptidesthat are differentially modified during or after translation, e.g., byphosphorylation, glycosylation, crosslinking, acylation, proteolyticcleavage, linkage to an antibody molecule, membrane molecule or otherligand, (Ferguson et al., Ann. Rev. Biochem. 57: 285-320, 1998).

New antigens and novel epitopes also can be identified using methodswell known in the art. Any conventional method, e.g., subtractivelibrary, comparative Northern and/or Western blot analysis of normal andtumor cells, Serial Analysis of Gene Expression (U.S. Pat. No.5,695,937) and SPHERE (described in PCT WO 97/3 5 03 5), can be used toidentify putative antigens for use.

For example, expression cloning as described in Kawakami et al., 1994,Proc. Natl. Acad. Sci. 91: 3515-19, also can be used to identify a noveltumor-associated antigen. Briefly, in this method, a library of cDNAscorresponding to mRNAs derived from tumor cells is cloned into anexpression vector and introduced into target cells which aresubsequently incubated with cytotoxic T cells. Pools of cDNAs that areable to stimulate T Cell responses are identified and through a processof sequential dilution and re-testing of less complex pools of cDNAs,unique cDNA sequences that are able to stimulate the T cells and thusencode a tumor antigen are identified. The tumor-specificity of thecorresponding mRNAs can be confirmed by comparative Northern and/orWestern blot analysis of normal and tumor cells.

SAGE analysis can be employed to identify the antigens recognized byexpanded immune effector cells such as CTLs, by identifying nucleotidesequences expressed in the antigen-expressing cells. SAGE analysisbegins with providing complementary deoxyribonucleic acid (cDNA) from anantigen-expressing population and cells not expressing the antigen. BothcDNAs can be linked to primer sites. Sequence tags are then created, forexample, using appropriate primers to amplify the DNA. By measuring thedifferences in these tag sets between the two cell types, sequenceswhich are aberrantly expressed in the antigen-expressing cell populationcan be identified.

Another method to identify optimal epitopes and new antigenic peptidesis a technique known as Solid PHase Epitope REcovery (“SPHERE”). Thismethod is described in detail in PCT WO 97/35035. Although used toscreen for MHC class I— restricted CTL epitopes, the method can bemodified to screen for class II epitopes by screening for thestimulation of antigen-specific MHC class II specific T cell lines, forexample, rather than CTL. In SPHERE, peptide libraries are synthesizedon beads where each bead contains a unique peptide that can be releasedin a controlled manner. Eluted peptides can be pooled to yield wellswith any desired complexity. After cleaving a percentage of the peptidesfrom the beads, these are assayed for their ability to stimulate a ClassII response, as described above. Positive individual beads are then bedecoded, identifying the reactive-amino acid sequence. Analysis of allpositives will give a partial profile of conservatively substitutedepitopes which stimulate the T cell response being tested. The peptidecan be resynthesized and retested to verify the response. Also, a secondlibrary (of minimal complexity) can be synthesized with representationsof all conservative substitutions in order to enumerate the completespectrum of derivatives tolerated by a particular response. By screeningmultiple T cell lines simultaneously, the search for crossreactingepitopes can be facilitated.

Isolated peptides can be synthesized using an appropriate solid statesynthetic procedure (Steward and Young, Solid Phase Peptide Synthesis,Freemantle, San Francisco, Calif. 1968). A preferred method is theMerrifield process (Merrifield, Recent Progress in Hormone Res. 23: 451,1967). The antigenic activity of these peptides may conveniently betested using, for example, the assays as described herein.

Once an isolated peptide is obtained, it may be purified by standardmethods including chromatography (e.g., ion exchange, affinity, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for protein purification. Forimmunoaffinity chromatography, an epitope may be isolated by binding itto an affinity column comprising antibodies that were raised againstthat peptide, or a related peptide, and were affixed to a stationarysupport. Alternatively, affinity tags such as hexa-His (Invitrogen),Maltose binding domain (New England Biolabs), influenza coat sequence(Kolodziej, et al., Methods Enzymol. 194: 508-509, 1991), andglutathione-S-transferase can be attached to the peptides to allow easypurification by passage over an appropriate affinity column. Isolatedpeptides also can be physically characterized using such techniques asproteolysis, nuclear magnetic resonance, and x-ray crystallography.

Having isolated and identified the peptide sequence of a desiredepitope, nucleic acids comprising sequences encoding these epitopes canbe sequenced readily.

Endocytic Trafficking Sequences

The available data suggest the following sequence of events in theintracellular transport of MHC class II molecules: MHC class IImolecules with the invariant chain are assembled in the endoplasmicreticulum and transported through the Golgi in common with othermembrane proteins including MHC class I. The molecules are then targetedto specific endosomal/lysosomal organelles by an unknown mechanism,segregating from the MHC class I molecules which follow a constitutiveroute to the cell surface. In the endocytotic/lysosomal route, theinvariant chain is removed from MHC class II by proteases acting in anacidic environment. At the same time, antigenic fragments of proteinsthat have entered the endocytic/lysosomal pathway are generated by theseproteases and the resulting peptides bind to the class II molecules andare carried to the cell surface.

The biosynthesis and vacuolar targeting mechanisms of the hydrolyticenzymes present in the lysosomal/endosomal compartment have beenextensively studied (Kornfeld and Mellman, Ann. Rev. Cell Biol. 5: 483,1989). Newly synthesized hydrolases in the Golgi apparatus acquiremannose 6-phosphate groups that serve as specific recognition markersfor the binding of these enzymes to mannose 6-phosphate receptors whichare then targeted in some unknown manner to a prelysosomal vacuole.There the receptor-enzyme complex is dissociated by low pH, and thereceptors recycle to the Golgi apparatus, while the enzyme-containingvacuole matures into a lysosome.

The localization of the lysosomal membrane glycoproteins is controlledby a targeting mechanism independent of the well-defined mannose6-phosphate receptor (MPR) pathway for hydrolytic lysosomal enzymes(Kornfeld and Mellman, 1989, supra). Recent studies describe a distinctvesicular compartment with lysosomal properties and characterized byhigh concentration of lysosomal-associated membrane protein (LAMP-1) andMHC class II molecules (Peters, et al., EMBO J. 9: 3497, 1990).Lysosomal/Endosomal Compartment Kinetic analysis of intracellulartransport and targeting of newly synthesized LAMP-1 and other similarproteins indicate that the molecule is synthesized in the endoplasmicreticulum, processed in the Golgi cisternae and transported to lysosomeswithin one hour of its biosynthesis, without detectable accumulation inthe plasma membrane (Barriocanal, et al., J Biol. Chem. 15: 261(35):16755-63, 1986; D'Sousa, et al., Arch. Biochem. Biophys. 249: 522, 1986;Green, et al., J. Cell Biol., 105: 1227, 1987).

Studies of the structure and function of the lysosomal membrane wereinitiated in 1981 by August and colleagues with the discovery of majorcellular glycoproteins that were subsequently termedlysosomal-associated membrane proteins one and two (LAMP-1 and LAMP-2)due to their predominant localization in the lysosomal membrane (Hughes,et al., J. Biol. Chem. 256: 664, 1981; Chen, et al., J. Cell Biol.101:85, 1985). Analogous proteins were subsequently identified in rat,chicken and human cells (Barriocanal, et al., 1986, supra; Lewis, etal., J. Cell Biol. 100:1839, 1985; Fambourgh, et al., J. Cell Biol. 106:61, 1988; Mane, et al., Arch. Biochem. Biophys. 268: 360, 1989).

Typically, LAMP-1, as deduced from a cDNA clone (Chen, et al., J. Biol.Chem. 263: 8754, 1988) consists of a polypeptide core of about 382 aminoacids with a large (346-residue) lumenal amino-terminal domain followedby a 24-residue hydrophobic transmembrane region and short (12-residue)carboxyl-terminal cytoplasmic tail. The lumenal domain is highlyglycosylated, being substituted with about 20 asparagine linkedcomplex-type oligosaccharides and consists of two to about 160-residuehomology units that are separated by a proline/serine-rich region. Eachof these homologous domains contains 4 uniformly spaced cysteineresidues, disulfide bonded to form four 36-38-residue loopssymmetrically placed within the two halves of the lumenal domain(Arterburn, et al., J. Biol. Chem. 265: 7419, 1990; see, also Chen, etal., J. Biol. Chem. 25: 263(18): 8754-8, 1988). The LAMP-2 molecule ishighly similar to LAMP-1 in overall amino acid sequence (Cha, et al., J.Biol. Chem. 265: 5008, 1990).

LAMP-1 and LAMP-2 are not specifically found in antigen presenting cells(dendritic cells). Their precise function is unknown, but theypresumably are involved in some manner with lysosome function. Theircolocalization with MHC II in the multilaminar MIIC vesicularcompartment of APCs has no known functional association to antigenprocessing or presentation; however, chimeric antigens comprising LAMPcytoplasmic domains, as discussed above, show enhanced immunogenicity(see, U.S. Pat. No. 5,633,234).

The invention provides chimeric proteins comprising the lumenal domainof a lysosomal associated membrane protein, such as a LAMP polypeptide,or a bioactive fragment or modified form thereof (collectively referredto as “a LAMP-lumenal domain”). In one aspect, the LAMP lumenal domaincomprises at least two homology units. Preferably, each homology unit isseparated by a proline/serine-rich region. More preferably, eachhomology domain comprises 4 cysteine residues capable of forming four36-38-residue loops symmetrically placed within the two halves of thelumenal domain when disulfide bonded together. Most preferably, thelumenal domain comprises sequences necessary to target and traffic apolypeptide to which it is linked (e.g., via a chemical bond) to anendosomal/lysosomal compartment or lysosome-related organelle forbinding to an MHC class II molecule or for delivery to anothercompartment/organelle where it will bind to an MHC class II molecule.

In another aspect, the chimeric protein additionally, or alternatively,comprises a dileucine-based signal comprising at least oneleucine-leucine pair or at least one leucine/isoleucine pair.Preferably, the protein further comprises an acidic residue 4-5 residuesupstream of the pair. More preferably, this signal domain binds to an APcomplex polypeptide (see, e.g., Bonafacino and Dell'Angelica, J. CellBiol. 145: 923-926, 1999). Suitable dileucine-based domains can be foundin tyrosinase (TM-X₁₀-EKQPLL-X₅-YHSL-X₅) (SEQ ID NO: 1); TRP-2(TM-X₇-EANQPLL-X₁₂) (SEQ ID NO: 2); and Pme17 (TM-X₃₄-ENSPLL-X₅) (SEQ IDNO: 3) and P-protein (see, e.g., Dell'Angelica, 2000, supra), forexample.

In a preferred aspect, the chimeric protein also comprises a cytoplasmicdomain for targeting and/or trafficking a chimeric protein to anendosomal/lysosomal compartment or lysosome-related organelle. In oneaspect, the cytoplasmic domain comprises the tail of a LAMP polypeptide.The eleven amino-acid sequence of the cytoplasmic tail of LAMP-1 andother similar lysosomal membrane glycoproteins has the followingsequence: Arg-Lys-Arg-Ser-His-Ala-Gly-Tyr-Gln-Thr-Ile-COOH (SEQ ID NO:4) (Chen, et al., 1988, supra). In LAMP-1, these sequences are fromamino acids 372-382 of the full-length polypeptide.

The known cytoplasmic tail sequences of lysosomal membrane proteins,LAMP-1 (Chen, et al., 1988, supra), LAMP-2 (Cha, et al., 1990, supra)and CD63 (Hotta, et al., Cancer Res. 48: 2955, 1988), have been alignedby the inventors with the Tyr-containing internalization signal in thecytoplasmic tail of LAP (Pohlman, et al., EMBO J. 7: 2343, 1988) inTable 1. The Tyr residue is known to be required for endosomal/lysosomaltargeting, and it was demonstrated in U.S. Pat. No. 5,633,234 that thecomplete sequence required to target other molecules to lysosomesrequires the Tyr-X-X-hyd sequence (i.e., a “Tyr motif”), a Tyr followedby two amino acids followed by a hydrophobic residue.

TABLE 1Cytoplasmic Tail Sequences of the Major Lysosomal Membrane Proteins*LAMP-1: R K R S H A G Y Q T I(SEQ ID NO: 4) LAMP-2:K H H A     G Y E Q F(SEQ ID NO: 5) CD63:K S I R S   G Y E V M(SEQ ID NO: 6) LAP:R M E A P P G Y R H V A D G Q D H A(SEQ ID NO: 7) *The conservedGly-Tyr-X-X-hydrophobic residue motif in the cytoplasmic domain of thedescribed lysosomal membrane proteins is underlined, where X is anyamino acid. The complete cytoplasmic tail sequence of the listedproteins is shown from the transmembrane region to the carboxylterminus.

The importance of a hydrophobic residue at or near the carboxyl-terminalposition is shown by results obtained from modification of theTyr-Gln-Thr-Ile (SEQ ID NO: 8) sequence of LAMP-1. Mutant cDNA moleculesin which Be was substituted with two other hydrophobic residues, Leu orPhe, and a polar residue, Thr. Substituting Leu (Tyr-Gln-Thr-Leu) (SEQID NO: 9) and Phe (Tyr-Gln-Thr-Phe) (SEQ ID NO: 10) does not affectlysosomal targeting, whereas the Thr-containing mutant protein(Tyr-Gln-Thr-Thr) (SEQ ID NO: 11) accumulates at the cell surface.Mutants containing Ala substituted for Gln (Tyr-Ala-Thr-Ile) (SEQ ID NO:12), Thr (Tyr-Gln-Ala-Ile) (SEQ ID NO: 13), and both residues(Tyr-Ala-Ala-Ile) (SEQ ID NO: 14) have no effect on targeting to thelysosomal membrane, indicating that these positions may be occupied bycharged, polar, or nonpolar residues.

The preferred targeting signal to the lysosomal/endosomal compartment,therefore, includes a tetrapeptide sequence located in the cytoplasmicdomain, near the transmembrane domain and also near the C-terminus. Thecytoplasmic domain is preferably a short amino acid sequence (less than70 amino acids, preferably less than 30 amino acids, most preferablyless than 20 amino acids) ending in a free carboxyl group. In a morepreferred embodiment, the tetrapeptide is at the C-terminal end of ashort cytoplasmic tail that contains the targeting signal, or is in acontext similar to LAMP-1.

A suitable four amino acid sequence for the tetrapeptide may be obtainedby amino acid substitutions, so long as the motif consists ofTyr-X-X-Hyd (where X may be any amino acid and Hyd denotes a hydrophobicamino acid), and the ability to confer lysosomal/endosomal targeting isconserved. A particularly preferred tetrapeptide has the sequenceTyr-Gln-Thr-Ile (SEQ ID NO: 8). In the most preferred embodiment, theentire LAMP cytoplasmic tail in conjunction with its transmembranedomain, and most preferably, its luminal domain is coupled to theprimary sequence of the antigenic domain for highly efficient MHC classII processing and presentation. However, the cytoplasmic domain is notnecessary to facilitate trafficking so long as a lumenal domain of aLAMP polypeptide is provided.

In another aspect, the endosomal targeting domain comprises atransmembrane sequence. Many proteins that will serve as the source ofthe antigenic domain for particular immune stimulatory constructs willbe surface antigens that include a transmembrane domain in their primarysequence. Such a transmembrane domain can be retained, and thecytoplasmic domain replaced with a lysosomal/endosomal targeting domainas taught herein (e.g., a domain comprising a LAMP lumenal domain).

In one preferred aspect, the transmembrane domain of LAMP (see, Chen, etal., J. Biol. Chem. 263: 8754, 1988) is coupled to the primary sequenceof a desired antigenic domain and the sequence of the lumenal domain.The structure of a transmembrane domain in a polypeptide is well knownin the art (see, e.g., Bangham, Anal. Biochem. 174: 142, 1988; Klein, etal., Biochem. Biophys. Acta 815: 468, 1985; Kyle & Doolittle, J. Mol.Biol. 157: 105, 1982). Usually the transmembrane region appears in theprimary sequence as a sequence of 20-25 hydrophobic amino acid residuesflanked by more hydrophilic regions.

Such sequences can be found, for example, in most cell surface antigensequences listed by Genebank as well as many other membrane proteins.The particular transmembrane sequence is not critical, so long as itserves to connect the antigenic domain to the lumenal domain andcytoplasmic tail and anchors the construct in the membranouscompartment.

Additional, or alternative sorting motifs, can include, but are notlimited to, one or more of: a targeting domain, a tyrosine motif domainas described above; a di-leucine and tyrosine-based domain; a prolinerich domain; and S-V-V domain (see, e.g., Blott and Grifitts, Nature 3:122-131, 2002).

Endocytic Receptor Sequences

Antigen access to the MHC II vesicular compartment of antigen presentingcells, such as dendritic cells, is normally by endocytosis of foreignantigens. It is the discovery of the instant invention that thetrafficking domains of endocytic receptors can be used to generatechimeric polypeptides to carry antigens to endosomal/lysosomalcompartments or to lysosome-related organelles for association withclass II MHC molecules and subsequent processing.

In one aspect, therefore, the invention provides an antigen linked to atrafficking domain of an endocytic receptor (e.g., via in-frame fusionof nucleic acid sequences encoding the trafficking domain and antigen).The trafficking domain localizes the antigen to an endosomal/lysosomalcompartment or to a lysosome-related organelle for association with anMHC class II molecule in the compartment/organelle or in a subsequentcompartment to which the antigen is delivered.

Endocytic receptors according to the invention, include, but are notlimited to receptors for microorganisms, Fc receptors (e.g., CD64, CD32,CD16, CD23, and CD89); complement receptors (e.g., CR1 or CD35, CR3,CR4); scavenger receptors or receptors which bind to acetylated ormodified lipoproteins, polyribonucloetides, lipopolysaccharides andsilica particles (e.g., such as SRA, MARCO), integrins (CD49e/CD29;CD49d/CD29; CD51/CD61); lectins (e.g., such as dectin-1, C-type lectins,and the like), and Toll-like receptors (e.g., TLRs). For a review ofsuch receptors, see Underhill and Ozinsky, Annu. Rev. Immunol. 20:825-52, 2002, for example.

In one aspect, the endocytic receptor is obtained from a professionalantigen presenting cell such as a dendritic cell. A number of endocyticreceptors of dendritic cells have been identified, including themacrophage mannose receptor (MMR), phospholipaseA₂-receptor, Endo 180,and DEC-205 and its human homologue, gp200-MR6 (McKay, et al., 1998).DEC-205 is reported to differ from the MMR, at least, in that it targetsantigenic material to an endosomal/lysosomal compartment co-localizedwith LAMP and MHC II, whereas MMR is found in peripheral endosomeslacking LAMP and MHC II (Mahnke, et al., J. Cell Biol. 151(3): 673-684,2000).

DEC-205 also demonstrates a greatly enhanced presentation of endocytosedantigen to CD4⁺ T-cells, as compared to that by the MMR. This differencein trafficking and antigen delivery to MH II between the two moleculesis reported to result from the presence in the cytosolic tail ofDEC-205, in addition to the coated pit uptake sequence, of an EDE triadthat is lacking in the MMR. The distal portion of the cytosolic tailcontaining the EDE sequence was shown to be required for the targetingto the deeper endosome/lysosome compartment containing LAMP and MHC II,and EDE was not replaced by an AAA sequence. Mahnke et al., 2000, supra,have also shown that these cytoplasmic tail trafficking signals aresufficient to traffic and recycle a CD 16 chimera to the MHC II/LAMPsite and to mediate a 100-fold increase in antigen presentation.

The sequence similarity between DEC-205 and gp200-MR6, particularly, inthe cytoplasmic domain, makes this sequence a suitable traffickingsequence as well. Further, gp200-MR6 has been shown to have the furtherimportant property of IL-4 regulation. McKay et al., Eur J. Immunol.28(12): 4071-83, 1998, have shown that ligation of gp200-MR6 can mimicIL-4 and have an antiproliferative, pro-maturational influence withinthe immune system, causing up-regulation of costimulatory molecules on Blymphocytes.

It is a discovery of the instant invention, however, that DEC-205fusions with LAMP do not traffic to the endosomal compartment but ratherlocalize to the cell surface. Chimeric proteins which combine LAMPdomains, an antigen sequence containing at least one epitope, andendocytic receptor domains such as DEC-205 domains, however, are able totraffic to endosomal compartments, co-localizing with endogenous LAMP.

Therefore, in a preferred aspect, a chimeric protein of the inventioncomprises a lumenal domain of a lysosomal membrane polypeptide (e.g.,such as a LAMP lumenal domain) and the targeting domain of an endocyticreceptor (e.g., such as DEC-205 or gp 200-MR6 polypeptide). Suchconstructs not only show correct targeting but improved antigenicity aswell. In a further aspect, both the targeting and trafficking domain ofan endocytic receptor is provided along with the antigen domaincomprising at least one epitope. Chimeric proteins may additionally, oralternatively, comprise the lumenal domain of an endocytic receptor. Instill a further aspect, a chimeric protein may comprise a full-lengthendocytic receptor polypeptide along with the antigenic domaincomprising at least one epitope.

In one aspect, the targeting domain of the endocytic receptor comprisesthe 31 amino acid cytoplasmic domain of a DEC polypeptide (see, e.g.,Manhke, et al., 2000, supra). In another aspect, the targeting domaincomprises residues 7-9 of the DEC-205 cytoplasmic tail. Preferably, thedomain comprises a Tyr motif. More preferably, the targeting domain alsocomprises residues 18-27 of the DEC-205 cytoplasmic tail. In a furtheraspect, the targeting domain comprises an EDE domain. The sequence ofDEC-205 is provided in Kato, et al., Immunogenetics 47(6): 442-50, 1998,while that of gp200-MR6 is provided in McKay, et al., 1998, supra, forexample.

The chimeric protein may additionally comprise a cytoplasmic targetingdomain for targeting a polypeptide to a endosomal/lysosomal compartmentor a lysosome-related organelle (e.g., such as a cytoplasmic LAMPdomain) as well as one or more of the other domains described above(e.g., signal sequence, transmembrane sequence, etc.). As above,additional, or alternative sorting motifs, can include, but are notlimited to, one or more of the M6P domain; a tyrosine motif domain; adi-leucine and tyrosine-based domain; a proline rich domain; and S-V-Vdomain (see, e.g., Blott and Grifitts, Nature 3: 122-131, 2002).

Vaccine Compositions

Tumors re-express developmental or embryonic genes which are notexpressed in normal cells in the individual. The major thrust of cancerimmunotherapy is the identification of tumor specific antigens and thedevelopment of immunization strategies that will most effectivelygenerate T cell dependent immunity against these antigens. For example,studies indicate that vaccinia virus recombinant vaccines containingeither the SV40 T antigen genes or the E6 and E7 genes from HPV orinfluenza nucleoprotein will protect animals against subsequentchallenges with tumor cells that express these proteins as tumorantigens. The protection is associated with the generation of antigenspecific responses among T cells in host.

A number of genes encoding tumor-specific antigenic polypeptides havebeen identified. Such antigens include, but are not limited to a tumorantigen, e.g., a polypeptide comprising an epitope derived from gp 100,MAGE 1, MART, MUC I and tyrosinase-related-protein 1 and 2 (TRP-1,TRP-2) (see, e.g., Boon et al., Immunol. Today 16: 334-336, 1998). MARTIand gp 100 are melanocyte differentiation antigens specificallyrecognized by HLA-A2 restricted tumor-infiltrating lymphocytes (TILs)derived from patients with melanoma, and appear to be involved in tumorregression (Kawakami et al., Proc. Natl. Acad. Sci. USA 91:645 8-62,1994; Kawakami, et al., Proc. Natl. Acad. Sci. USA 91: 3515-9).

EBV Epstein-Ban virus gene products also encode antigenic polypeptideswhich are expressed in Hodgkin's lymphomas as well as Burkits and otherlymphomas. Products of the HTLV-1 genome have been found in adult T cellleukemia cells, while human papillomavirus (HPV) E6 and E7 gene productsare found in cervical carcinoma cells.

Differential screening of nucleic acid sequences expressed by the twocell lines can be used to select sequences encoding antigens specific tocancer cells, and even specific stages of cancer cells. When thenon-target cell is a normal cell, differential screening eliminates orreduces the nucleic acid sequences common to normal cells, therebyavoiding an immune response directed at antigens present on normalcells. When the non-target cell is a normal cell, differential screeningeliminates or reduces sequences common to normal cells, thereby avoidingan immune response directed at antigens present on normal cells.

In many cases, it has been demonstrated that peptides derived fromaltered genetic sequences can associate with either MHC class I or MHCclass II molecules and be recognized by the appropriate helper orcytotoxic T cells. Mutations in various oncogenes such as the position12 mutation in K ras have been implicated as a major genetic alterationof colon cancer as well as other malignancies. Mutations in tumorsuppressor genes, such as P53, are extremely common in manymalignancies. Additionally, rearrangements that result in activation ofoncogenes such as the rearrangement between the BCR and abl gene inchronic myelogenous leukemia generate novel protein sequences.Therefore, in one preferred aspect of the invention, the antigenicmaterial used comprises a cancer-specific polypeptide or an alteredpolypeptide sequence (e.g., derived from a genetic mutation and/orchromosomal mutation) or which is more generally associated with anabnormal physiological response (e.g., an autoimmune response, ahypersensitivity reaction, a reaction to a transplant or graft, and thelike).

Any strategy which would enhance the presentation of a particularantigen on MHC molecules of host antigen presenting cells would, infact, enhance the immunization potential of such a viral based strategyfor a disease such as cancer, or another abnormal physiologicalresponse. The equivalent arguments can be made for generation ofenhanced vaccine efficacy for viral infections such as HIV.

Therefore, in one embodiment, this invention provides a vaccinecomposition for eliciting an immune response in a mammal to an antigen.The composition comprises a vaccine vector which comprises a chimericDNA segment comprising a sequence encoding at least one epitope of anantigen. In one aspect, the sequence encoding the antigen is from thelumenal domain of a protein. Preferably, the DNA segment furtherincludes a sequence encoding a lumenal domain of a lysosome associatedmembrane polypeptide (e.g., such as a LAMP polypeptide, homolog,ortholog, variant, or modified form thereof) or the trafficking domainof an endocytic receptor. Preferably, the lumenal domain or traffickingdomain traffics the antigen to an endosomal/lysosomal compartment or toa lysosome-related organelle of a cell, where it binds to an MHC classII molecule or is processed for delivery to anothercompartment/organelle where it will subsequently bind to an MHC class IImolecule. More preferably, the antigen is processed within thecompartment/organelle (or subsequent compartment to which it isdelivered) to generate an epitope which is presented on the surface ofthe cell and which is bound to the MHC class II molecule.

The vector also may encode one or more of a transmembrane domain, acytoplasmic domain containing an endosomal/lysosomal targeting signaldirecting the protein to an endosomal/lysosomal compartment orlysosome-related organelle, a dileucine domain, a Tyr motif; a prolinerich domain; and S-V-V domain.

The e domains may be provided in sequence or separated by nucleic acidsencoding linker polypeptides or which encode other amino acid sequenceswith desired functionalities (e.g., protein stabilizing sequences, andthe like). Generally, where linker sequences are included, these encodelinker polypeptides which range from about one to about 50 amino acids.The minimal requirement of the vector is that it encode a chimericprotein with the desired trafficking properties. Such properties can bereadily tested using assays routine in the art.

For example, immunofluorescence microscopy can be used to confirm thetrafficking of a chimeric protein to an appropriatecompartment/organelle. [³⁵S]methionine pulse-chase labeling analysis canbe used to monitor the synthesis and degradation of the chimeric proteinto demonstrate that the rates of synthesis of the chimeric protein vs.the endogenous protein comprising the antigen domain are essentiallyequal and/or that the processing of the chimeric protein occursproperly.

In particular embodiments, the protein encoded by the chimeric DNAsegment contains an intralumenal domain comprising at least one epitopewhich is a peptide that complexes with major histocompatibility complex(MHC) class II molecules, an endosomal/lysosomal trafficking sequence asdescribed above, and a cytoplasmic domain which contains anendosomal/lysosomal targeting sequence. Preferably, the targetingsequence comprises the tetrapeptide sequence Tyr-Xaa-Xaa-Xbb, whereinXbb is a hydrophobic amino acid.

In another aspect, the protein encoded by the chimeric DNA segmentcomprises a full length lysosomal membrane associated polypeptide, suchas a LAMP polypeptide, homolog, ortholog, variant or modified formthereof, which comprises sequences for targeting and trafficking bothmembrane-bound and non-membrane bound antigenic material to anendosomal/lysosomal compartment.

Recombinant Protein Chimera-Mediated Expression Of HIV-1 Gag Protein

In one aspect, the chimeric protein comprises an antigen from anHIV-encoded polypeptide. Induction of potent humoral and cellular immuneresponses, including those against viral structural genes have beenshown to be crucial to HIV virus clearance. Specific-CD4 anti-Gagresponses correlate with increased clinical resistance to the virus inHIV-infected patients and in a model of therapeutic vaccine inSW-infected macaques. Therefore, in one preferred aspect, the antigendomain of the chimeric protein comprises a Gag epitope.

Gag is relatively conserved among diverse HIV strains and subtypes andbroad cross-clad anti-Gag CTL responses have been demonstrated inHIV-infected patents. Studies of exposed but sero-negative subjectsindicate that Gag-specific CTL may be involved in protection against theestablishment of a persistant HIV infection. Additionally, Gag-specificCD8⁺ cytotoxic T lymphocytes are important in controlling virus loadduring acute infection as well as during the asymptomatic stages of theinfection. Multiple discrete Gag epitopes have been described and shownto mediate cytotoxic activities. Moreover, levels of p24-specific CTLproliferative responses of infected untreated persons were positivelycorrelated with levels of Gag-specific CTL and negatively correlatedwith levels of plasma HIV-1 RNA.

The expression of the human immunodeficiency virus type 1 (HIV-1) genewhen it is introduced into mammalian cells is tightly regulated at manylevels. At a post-transcriptional step, the binding of HIV-1 Rev to anelement of the Gag mRNA found in all unspliced and singly spliced HIV-1mRNAs, the Rev-responsive element (RRE), results in the nuclear exportof the mRNA and translation of Gag, Gag-Pol, Env, Vif, Vpr, and Vpu. Inthe absence of Rev, cells transfected with DNA encoding Gag producelittle or no the Gag protein. Rev may also act to stabilize HIV-1 mRNAsby its effect on the high AU content instability sequences and itseffect on certain cis-acting instability sequences (INS). (For a recentreport, see Korsopoulou et al, 2000, for example).

The dependence on Rev for expression of the Gag protein is a severeproblem for the use of this protein in the development of a Gag-basedvaccine against HIV-1 with DNA plasmid vectors because of therequirement to provide an accessory factor for nuclear export ofunspliced retroviral mRNA. Several procedures have been used by othersto overcome the Rev dependence. The HIV-1 genes have an unusual codonbias, differing markedly from that of human genes, and humanizing thegene sequence has been found to enhance the translation of HIV-1 mRNAs(Haas, et al, Curr Biol. 6(3): 315-24, 1996). Similarly, removal of theINS sequences increases the translational efficiency (Schneider et al, JVirol. 71(7): 4892-903, 1997). Others have also described certain cisactive constitutive transport elements (CTE) present in the non-codingregions of simial type D retroviruses and lentiviruses (for a reviewsee, Cullen, Virology 248: 203-210, 1998). This sequence folds into anextended RNA stem-loop structure which binds a transport associatedprotein (TAP) which promotes nucleocytoplasmic transport the mRNA.

In one aspect, the invention provides compositions to increase theprotein expression of HIV-1 gag in the absence of HIV-1 Rev, a geneproduct normally required for its roles in HIV-1 mRNA translation andnuclear export. This expression of gag in cells transfected with a HIV-1gag DNA plasmid is accomplished by synthesizing the gag gene as a DNAchimera encoding a gag sequence inserted into another highly expressedcellular protein. In one highly preferred aspect, the HIV-1 gag sequenceis inserted into the lumenal domain of a lysosomal membrane protein,preferably, adjacent to a trans-membrane domain. Such a chimera can beused to develop an anti-HIV-1 DNA vaccine comprising the gag gene.

DNA chimeric vaccines comprising nucleic acids encoding antigensequences combined with nucleic acids encoding the targeting domains ofan dendritic cell specific DEC-205 endocytic receptor and the lumenaltrafficking domains of LAMP can be produced. In one aspect, theendocytic receptor is a dendritic cell endocytic receptor, such asDEC-205. As shown in the examples below, DEC-205 fused to a LAMP lumenaldomain, HIV p55 gag, and the transmembrane and cytoplasmic tail ofDEC-205 showed co-localizarion of the HIV antigen provided as part ofthe chimeric protein with the endogenous LAMP protein. The LAMP/Gag/DECconstruct presented an equivalent immunogenicity to the LAMP/Gagconstruct.

Assembly of Sequences Encoding Chimeric Proteins

Procedures for construction of chimeric proteins are well known in theart (see e.g., Williams, et al., J. Cell Biol. 111: 955, 1990). DNAsequences encoding the desired segments can be obtained from readilyavailable recombinant DNA materials such as those available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, U.S.A., or from DNA libraries that contain the desired DNA.

Such DNA segments minimally include: sequences encoding an antigenicdomain and a lumenal domain of a lysosomal membrane associatedpolypeptide for trafficking a polypeptide linked to the lumenal domainto an endosomal/lysosomal compartment or lysosome-related organelleand/or a trafficking domain of an endocytic receptor for trafficking toan endosomal/lysosomal compartment and or lysosome-related organelle.Additional DNA segments may include, but are not limited to, sequencesencoding: cytoplasmic targeting sequences for targeting the chimericprotein to an endosomal/lysosomal compartment or lysosome-relatedorganelle; transmembrane sequences, signal sequences, di-leucinesequences, Tyr motifs, proline rich domains, M6P sequences, Ser-Val-Valsequences and the like.

The DNA segments corresponding to the desired domain sequences are thenassembled with appropriate control and signal sequences using routineprocedures of recombinant DNA methodology. See, e.g., as described inU.S. Pat. No. 4,593,002, and Langford, et al., Molec. Cell. Biol. 6:3191, 1986.

A DNA sequence encoding a protein or polypeptide can be synthesizedchemically or isolated by one of several approaches. The DNA sequence tobe synthesized can be designed with the appropriate codons for thedesired amino acid sequence. In general, one will select preferredcodons for the intended host in which the sequence will be used forexpression. The complete sequence may be assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature 292: 756, 1981;Nambair, et al. Science 223: 1299, 1984; Jay, et al., J. Biol. Chem.259: 6311, 1984.

In one aspect, one or more of the nucleic acids encoding the domainsequences of the chimeric protein are isolated individually using thepolymerase chain reaction (M. A. Innis, et al., In PCR Protocols: AGuide To Methods and Applications, Academic Press, 1990). The domainsare preferably isolated from publicly available clones known to containthem, but they may also be isolated from genomic DNA or cDNA libraries.Preferably, isolated fragments are bordered by compatible restrictionendonuclease sites which allow a chimeric DNA encoding the immunogenicprotein sequence to be constructed. This technique is well known tothose of skill in the art. Domain sequences may be fused directly toeach other (e.g., with no intervening sequences), or inserted into oneanother (e.g., where domain sequences are discontinuous), or mayseparated by intervening sequences (e.g., such as linker sequences).

The basic strategies for preparing oligonucleotide primers, probes andDNA libraries, as well as their screening by nucleic acid hybridization,are well known to those of ordinary skill in the art. See, e.g.,Sambrook, et al., 1989, supra; Perbal, 1984, supra. The construction ofan appropriate genomic DNA or cDNA library is within the skill of theart. See, e.g., Perbal, 1984, supra. Alternatively, suitable DNAlibraries or publicly available clones are available from suppliers ofbiological research materials, such as Clonetech and Stratagene, as wellas from public depositories such as the American Type CultureCollection.

Selection may be accomplished by expressing sequences from an expressionlibrary of DNA and detecting the expressed peptides immunologically.Clones which express peptides that bind to MHC II molecules and to thedesired antibodies/T cell receptors are selected. These selectionprocedures are well known to those of ordinary skill in the art (see,e.g., Sambrook, et al., 1989, supra).

Once a clone containing the coding sequence for the desired polypeptidesequence has been prepared or isolated, the sequence can be cloned intoany suitable vector, preferably comprising an origin of replication formaintaining the sequence in a host cell.

Nucleic Acid Delivery Vehicles

In one aspect, a nucleic acid vector encoding a chimeric vaccine isintroduced into a cell. The cell may be a host cell for replicating thenucleic acid or for expressing the chimeric vaccine. Preferably, thehost cell for expressing the chimeric vaccine is an antigen presentingcell (described further below).

The nucleic acid vector minimally comprises a polynucleotide sequencefor insertion into a target cell and an expression control sequenceoperably linked thereto to control expression of the polynucleotidesequence (e.g., transcription and/or translation) in the cell. Examplesinclude plasmids, phages, autonomously replicating sequences (ARS),centromeres, and other sequences which are able to replicate or bereplicated in vitro or in a host cell (e.g., such as a bacterial, yeast,or insect cell) and/or target cell (e.g., such as a mammalian cell,preferably an antigen presenting cell) and/or to convey the sequencesencoding the chimeric vaccine to a desired location within the targetcell.

Recombinant expression vectors may be derived from micro-organisms whichreadily infect animals, including man, horses, cows, pigs, llamas,giraffes, dogs, cats or chickens. Preferred vectors include those whichhave already been used as live vaccines, such as vaccinia. Theserecombinants can be directly inoculated into a host, conferring immunitynot only to the microbial vector, but also to express foreign antigens.Preferred vectors contemplated herein as live recombinant vaccinesinclude RNA viruses, adenovirus, herpesviruses, poliovirus, and vacciniaand other pox viruses, as taught in Flexner, Adv. Pharmacol. 21: 51,1990, for example.

Expression control sequences include, but are not limited to, promotersequences to bind RNA polymerase, enhancer sequences or negativeregulatory elements to bind to transcriptional activators andrepressors, respectively, and/or translation initiation sequences forribosome binding. For example, a bacterial expression vector can includea promoter such as the lac promoter and for transcription initiation,the Shine-Dalgarno sequence and the start codon AUG (Sambrook, et al.,1989, supra). Similarly, a eukaryotic expression vector preferablyincludes a heterologous, homologous, or chimeric promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of a ribosome.

Expression control sequences may be obtained from naturally occurringgenes or may be designed. Designed expression control sequences include,but are not limited to, mutated and/or chimeric expression controlsequences or synthetic or cloned consensus sequences. Vectors thatcontain both a promoter and a cloning site into which a polynucleotidecan be operatively linked are well known in the art. Such vectors arecapable of transcribing RNA in vitro or in vivo, and are commerciallyavailable from sources such as Stratagene (La Jolla, Calif.) and PromegaBiotech (Madison, Wis.).

In order to optimize expression and/or transcription, it may benecessary to remove, add or alter 5′ and/or 3′ untranslated portions ofthe vectors to eliminate extra, or alternative translation initiationcodons or other sequences that may interfere with, or reduce,expression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression. a wide varietyof expression control sequences—sequences that control the expression ofa DNA sequence operatively linked to it—may be used in these vectors toexpress the DNA sequences of this invention. Such useful expressioncontrol sequences include, for example, the early or late promoters ofSV40, CMV, vaccinia, polyoma, adenovirus, herpes virus and othersequences known to control the expression of genes of mammalian cells,and various combinations thereof.

In one aspect, the nucleic acid delivery vector comprises an origin ofreplication for replicating the vector. Preferably, the origin functionsin at least one type of host cell which can be used to generatesufficient numbers of copies of the sequence for use in delivery to atarget cell. Suitable origins therefore include, but are not limited to,those which function in bacterial cells (e.g., such as Escherichia sp.,Salmonella sp., Proteus sp., Clostridium sp., Klebsiella sp., Bacillussp., Streptomyces sp., and Pseudomonas sp.), yeast (e.g., such asSaccharamyces sp. or Pichia sp.), insect cells, and mammalian cells. Inone preferred aspect, an origin of replication is provided whichfunctions in the target cell into which the nucleic acid deliveryvehicle is introduced (e.g., a mammalian cell, such as a human cell).

In another aspect, at least two origins of replication are provided, onethat functions in a host cell and one that functions in a target cell.

The nucleic acid delivery vector may alternatively, or additionally,comprise sequences to facilitate integration of at least a portion ofthe nucleic acid deliver vector into a target cell chromosome. Forexample, the nucleic acid delivery vector may comprise regions ofhomology to target cell chromosomal DNA. In one aspect, the deliveryvector comprises two or more recombination sites which flank a nucleicacid sequence encoding the chimeric vaccine.

The vector may additionally comprise a detectable and/or selectablemarker to verify that the vector has been successfully introduced in atarget cell and/or can be expressed by the target cell. These markerscan encode an activity, such as, but not limited to, production of RNA,peptide, or protein, or can provide a binding site for RNA, peptides,proteins, inorganic and organic compounds or compositions and the like.

Examples of detectable/selectable markers genes include, but are notlimited to: DNA segments that encode products which provide resistanceagainst otherwise toxic compounds (e.g., antibiotics); DNA segments thatencode products which are otherwise lacking in the recipient cell (e.g.,tRNA genes, auxotrophic markers); DNA segments that encode productswhich suppress the activity of a gene product; DNA segments that encodeproducts which can be readily identified (e.g., phenotypic markers suchas β-galactosidase, a fluorescent protein (GFP, CFP, YFG, BFP, RFP,EGFP, EYFP, EBFP, dsRed, mutated, modified, or enhanced forms thereof,and the like), and cell surface proteins); DNA segments that bindproducts which are otherwise detrimental to cell survival and/orfunction; DNA segments that otherwise inhibit the activity of othernucleic acid segments (e.g., antisense oligonucleotides); DNA segmentsthat bind products that modify a substrate (e.g., restrictionendonucleases); DNA segments that can be used to isolate or identify adesired molecule (e.g., segments encoding specific protein bindingsites); primer sequences; DNA segments, which when absent, directly orindirectly confer resistance or sensitivity to particular compounds;and/or DNA segments that encode products which are toxic in recipientcells.

The marker gene can be used as a marker for conformation of successfulgene transfer and/or to isolate cells expressing transferred genesand/or to recover transferred genes from a cell. For example, in oneaspect, the marker gene is used to isolate and purify antigen presentingcells expressing the chimeric vaccines.

As discussed above, homologs, variants, and modified forms of any of thedomain sequences can be used so long as they retain the ability tofunction with their respective domain function. For example, a modifiedlumenal sequence must retain the ability to traffic both membrane andnon-membrane antigenic materials to an endosomal compartment with atleast about 50%, at least about 60%, at least about 80%, at least about90%, or at least about 100% efficacy compared to the original domainsequence, i.e., an efficacy that results in sufficient antigenpresentation by a cell comprising the chimeric sequence for it to mountan immune response. In one aspect, sequences containing a suitabletrafficking signal may be identified by constructing a chimeric DNAcontaining the well-characterized antigenic domain of ovalbumin, atransmembrane domain, and the cytoplasmic domain of a protein containinga putative lysosomal/endosomal targeting signal. Efficiency of targetingcan be measured by determining the ability of antigen presenting cells,expressing the chimeric protein, to stimulate HA epitope-specific, MHCclass II restricted T-cells (see, e.g., Example 5 of U.S. Pat. No.5,633,234).

Substantially similar genes may be provided, e.g., genes with greaterthan about 50%, greater than about 70%, greater than about 90%, andpreferably, greater than about 95% identity to a known gene. Percentidentity can be determined using software programs known in the art, forexample those described in Current Protocols In Molecular Biology (F. M.Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1.Preferably, default parameters are used for alignment. A preferredalignment program is BLAST, using default parameters. In particular,preferred programs are BLASTN and BLASTP, using the following defaultparameters: Genetic code=standard; filter=none; strand=both; cutoff=60;expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by ═HIGHSCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.govicgi-bin/BLAST.

“Conservatively modified variants” of genes also can be provided. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Specifically, degenerate codon substitutions can be achievedby generating sequences in which the third position of one or moreselected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res. 19: 5081;Ohtsuka, et al., 1985, J. Biol. Chem. 260: 2605-2608; Rossolini et al.,1994, Mol. Cell. Probes 8: 91-98).

Substantially similar domain sequences may initially be identified byselecting a sequence which specifically hybridizes to a domain sequenceof interest under stringent hybridization conditions. Examples ofstringent hybridization conditions include: incubation temperatures ofabout 25° C. to about 37° C.; hybridization buffer concentrations ofabout 6×SSC to about 10×SSC; formamide concentrations of about 0% toabout 25%; and wash solutions of about 6×SSC. Examples of moderatehybridization conditions include: incubation temperatures of about 40°C. to about 50° C.; buffer concentrations of about 9×SSC to about 2×SSC;formamide concentrations of about 30% to about 50%; and wash solutionsof about 5×SSC to about 2×SSC. Examples of high stringency conditionsinclude: incubation temperatures of about 55° C. to about 68° C.; bufferconcentrations of about 1×SSC to about 0.1×SSC; formamide concentrationsof about 55% to about 75%; and wash solutions of about 1×SSC, 0.1×SSC,or deionized water. In general, hybridization incubation times are from5 minutes to 24 hours, with 1, 2, or more washing steps, and washincubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and15 mM citrate buffer. It is understood that equivalents of SSC usingother buffer systems can be employed. Similarity can be verified bysequencing, but preferably, is also or alternatively, verified byfunction (e.g., ability to traffic to an endosomal compartment, and thelike), using assays suitable for the particular domain in question.

Performing assays to determine the suitability of homologous, variant,or modified domain sequences is merely a matter of screening forsequences which express the appropriate activity. Such screening isroutine in the art.

The nucleic acid delivery vector may be provided as naked nucleic acidsor in a delivery vehicle associated with one or more molecules forfacilitating entry of a nucleic acid into a cell. Suitable deliveryvehicles include, but are not limited to: liposomal formulations,polypeptides; polysaccharides; lipopolysaccharides, viral formulations(e.g., including viruses, viral particles, artificial viral envelopesand the like), cell delivery vehicles, and the like.

Lipid-Based Formulations

Delivery vehicles designed to facilitate intracellular delivery ofbiologically active molecules must interact with both non-polar andpolar environments (in or on, for example, the plasma membrane, tissuefluids, compartments within the cell, and the like). Therefore,preferably, delivery vehicles are designed to contain both polar andnon-polar domains or a translocating sequence for translocating anucleic acid into a cell.

Compounds having polar and non-polar domains are termed amphiphiles.Cationic amphiphiles have polar groups that are capable of beingpositively charged at, or around, physiological pH for interacting withnegatively charged polynucleotides such as DNA.

The nucleic acid vectors described above can be provided in formulationscomprising lipid monolayers or bilayers to facilitate transfer of thevectors across a cell membrane. Liposomes or any form of lipid membrane,such as planar lipid membranes or the cell membrane of an intact cell,e.g., a red blood cell, can be used. Liposomal formulations can beadministered by any means, including administration intravenously ororally.

Liposomes and liposomal formulations can be prepared according tostandard methods and are well known in the art, see, e.g., Remington's;Akimaru, 1995, Cytokines Mol. Ther. 1: 197-210; Alving, 1995, Immunol.Rev. 145: 5-31; Szoka, 1980, Ann. Rev. Biophys. Bioeng. 9: 467; U.S.Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; and U.S. Pat. No.4,837,028. In one aspect, the liposome comprises a targeting moleculefor targeting a liposome:nucleic acid vector complex to a particularcell type. In a particularly preferred aspect, a targeting moleculecomprises a binding partner (e.g., a ligand or receptor) for abiomolecule (e.g., a receptor or ligand) on the surface of a bloodvessel or a cell found in a target tissue.

Liposome charge is an important determinant in liposome clearance fromthe blood, with negatively charged liposomes being taken up more rapidlyby the reticuloendothelial system (Juliano, 1975, Biochem. Biophys. Res.Commun. 63: 651) and thus having shorter half-lives in the bloodstream.Incorporating phosphatidylethanolamine derivatives enhances thecirculation time by preventing liposomal aggregation. For example,incorporation of N-(omega-carboxy)acylamidophosphatidylethanolaminesinto large unilamellar vesicles of L-alpha-distearoylphosphatidylcholinedramatically increases the in vivo liposomal circulation lifetime (see,e.g., Ahl, 1997, Biochim. Biophys. Acta 1329: 370-382). Liposomes withprolonged circulation half-lives are typically desirable for therapeuticand diagnostic uses. For a general discussion of pharmacokinetics, see,e.g., Remington's, Chapters 37-39, Lee, et al., In PharmacokineticAnalysis: A Practical Approach (Technomic Publishing AG, Basel,Switzerland 1996).

Typically, liposomes are prepared with about 5 to 15 mole percentnegatively charged phospholipids, such as phosphatidylglycerol,phosphatidylserine or phosphatidyl-inositol. Added negatively chargedphospholipids, such as phosphatidylglycerol, also serve to preventspontaneous liposome aggregation, and thus minimize the risk ofundersized liposomal aggregate formation. Membrane-rigidifying agents,such as sphingomyelin or a saturated neutral phospholipid, at aconcentration of at least about 50 mole percent, and 5 to 15 molepercent of monosialylganglioside can also impart desirably liposomeproperties, such as rigidity (see, e.g., U.S. Pat. No. 4,837,028).

Additionally, the liposome suspension can include lipid-protectiveagents which protect lipids against free-radical and lipid-peroxidativedamages on storage. Lipophilic free-radical quenchers, such asalpha-tocopherol and water-soluble iron-specific chelators, such asferrioxianine, are preferred.

The nucleic acid delivery vehicles of the invention can includemultilamellar vesicles of heterogeneous sizes. For example,vesicle-forming lipids can be dissolved in a suitable organic solvent orsolvent system and dried under vacuum or an inert gas to form a thinlipid film. If desired, the film can be redissolved in a suitablesolvent, such as tertiary butanol, and then lyophilized to form a morehomogeneous lipid mixture which is in a more easily hydrated powderlikeform. This film is covered with an aqueous solution of the peptide orpolypeptide complex and allowed to hydrate, typically over a 15 to 60minute period with agitation. The size distribution of the resultingmultilamellar vesicles can be shifted toward smaller sizes by hydratingthe lipids under more vigorous agitation conditions or by addingsolubilizing detergents such as deoxycholate. The hydration mediumpreferably comprises the nucleic acid at a concentration which isdesired in the interior volume of the liposomes in the final liposomesuspension.

Following liposome preparation, the liposomes can be sized to achieve adesired size range and relatively narrow distribution of liposome sizes.One preferred size range is about 0.2 to 0.4 microns, which allows theliposome suspension to be sterilized by filtration through aconventional filter, typically a 0.22 micron filter. Filtersterilization can be carried out on a high throughput basis if theliposomes have been sized down to about 0.2 to 0.4 microns. Severaltechniques are available for sizing liposome to a desired size (see,e.g., U.S. Pat. No. 4,737,323).

Suitable lipids include, but are not limited to, DOTMA (Felgner, et al.,1987, Proc. Natl. Acad. Sci. USA 84: 7413-7417), DOGS or Transfectain™(Behr, et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6982-6986), DNERIEor DORIE (Felgner, et al., Methods 5: 67-75), DC-CHOL (Gao and Huang,1991, BBRC 179: 280-285), DOTAPTM (McLachlan, et al., 1995, Gene Therapy2: 674-622), Lipofectamine® and glycerolipid compounds (see, e.g.,EP901463 and W098/37916).

Other molecules suitable for complexing with nucleic acid deliveryvectors include cationic molecules, such as, polyamidoamine (Haenslerand Szoka, 1993, Bioconjugate Chem. 4: 372-379), dendritic polysine (WO95/24221), polyethylene irinine or polypropylene h-nine (WO 96/02655),polylysine (U.S. Pat. No. 5,595,897; FR 2 719 316), chitosan (U.S. Pat.No. 5,744,166), DNA-gelatin coarcervates (see, e.g., U.S. Pat. No.6,207,195; U.S. Pat. No. 6,025,337; U.S. Pat. No. 5,972,707) or DEAEdextran (Lopata, et al., 1984, Nucleic Acid Res. 12: 5707-5717).

Viral-Based Gene Delivery Vehicles

In one aspect, the nucleic acid delivery vehicle comprises a virus orviral particle. In this aspect, preferably, the nucleic acid vectorcomprises a viral vector. Viral vectors, such as retroviruses,adenoviruses, adeno-associated viruses and herpes viruses, are oftenmade up of two components, a modified viral genome and a coat structuresurrounding it (see, e.g., Smith et al., 1995, Ann. Rev. Microbiol. 49:807-838), although sometimes viral vectors are introduced in naked formor coated with proteins other than viral proteins. Most current vectorshave coat structures similar to a wild-type virus. This structurepackages and protects the viral nucleic acid and provides the means tobind and enter target cells.

Preferably, viral vectors are modified from wild-type viral genomes todisable the growth of the virus in a target cell while enabling thevirus to grow in a host cell (e.g., such as a packaging or helper cell)used to prepare infectious particles. Vector nucleic acids generallyessential cis-acting viral sequences for replication and packaging in ahelper line and expression control sequences for regulating theexpression of a polynucleotide being delivered to a target cell. Otherviral functions are expressed in trans in specific packaging or helpercell lines as are known in the art.

Preferred vectors are viral vectors derived from a virus selected fromthe group consisting of herpes viruses, cytomegaloviruses, foamyviruses, lentiviruses, Semliki forrest virus, AAV (adeno-associatedvirus), poxviruses, adenovirases and retroviruses. Such viral vectorsare well known in the art.

In one preferred aspect, a viral vector used is an adenoviral vector.The adenoviral genome consists of a linear double-stranded DNA moleculeof approximately 36 kb carrying more than about thirty genes necessaryto complete the viral replication cycle. The early genes are dividedinto 4 regions (E1 to E4) that are essential for viral replication withthe exception of the E3 region, which is believed to modulate theanti-viral host immune response. The E1 region (ElA and EIB) encodesproteins responsible for the regulation of transcription of the viralgenome. Expression of the E2 region genes (E2A and E2B) leads to thesynthesis of the polypeptides needed for viral replication. The proteinsencoded by the E3 region prevent cytolysis by cytotoxic T cells andtumor necrosis factor (Wold and Gooding, 1991, Virology 184: 1-8). Theproteins encoded by the E4 region are involved in DNA replication, lategene expression and splicing and host cell shut off (Halbert, et al.,1985, J. Virol. 56: 250-257). The late genes generally encode structuralproteins contributing to the viral capsid. In addition, the adenoviralgenome carries at cis-acting 5′ and 3′ ITRs (Inverted Terminal Repeat)and packaging sequences essential for DNA replication. The ITRs harbororigins of DNA replication while the encapsidation region is requiredfor the packaging of adenoviral DNA into infectious particles.

Adenoviral vectors can be engineered to be conditionally replicative(CRAd vectors) in order to replicate selectively in specific cells(e.g., such as proliferative cells) as described in Heise and Kim (2000,J. Clin. Invest. 105: 847-85 1). In another aspect, an adenoviral vectoris replication-defective for the E1 function (e.g., by total or partialdeletion or mutagenesis of E1). The adenoviral backbone of the vectormay comprise additional modifications (deletions, insertions ormutations in one or more viral genes). An example of an E2 modificationis illustrated by the thermosensitive mutation localized on the DBP (DNABinding Protein) encoding gene (Ensinger et al., 1972, J. Virol. 10:328-339). The adenoviral sequence may also be deleted of all or part ofthe E4 region (see, e.g., EP 974 668; Christ, et al., 2000, Human GeneTher. 11: 415-427; Lusky, et al., 1999, J. Virol. 73: 8308-8319).Additional deletions within the non-essential E3 region may allow thesize of the polynucleotide being delivered to be increased (Yeh, et al.,1997, FASEB Journal 11: 615 623). However, it may be advantageous toretain all or part of the E3 sequences coding for polypeptides (e.g.,such as gp19k) allowing the virus to escape the immune system (Gooding,et al., 1990, Critical Review of Immunology 10: 53-71) or inflammatoryreactions (EP 00440267.3).

Second generation vectors retaining the ITRs and packaging sequences andcomprising substantial genetic modifications to abolish the residualsynthesis of the viral antigens also may be used in order to improvelong-term expression of the expressed gene in the transduced cells (see,e.g., W0 94/28152; Lusky, et al., 1998, J. Virol 72: 2022-2032).

The polynucleotide being introduced into the cell may be inserted in anylocation of the viral genome, with the exception of the cis-actingsequences. Preferably, it is inserted in replacement of a deleted region(E1, E3 and/or E4), preferably, within a deleted E1 region.

Adenoviruses can be derived from any human or animal source, inparticular canine (e.g. CAV-1 or CAV-2 Genbank ref. CAVIGENOM andCAV77082, respectively), avian (Genbank ref. AAVEDSDNA), bovine (such asBAV3; Reddy, et al., 1998, J. Virol. 72: 1394 1402), murine (Genbankref. ADRMUSMAVI), ovine, feline, porcine or simian sources oralternatively, may be a hybrid virus. Any serotype can be employed.However, the human adenoviruses of the C sub-group are preferred,especially adenoviruses 2 (Ad2) and 5 (Ad5). Such viruses are available,for example, from the ATCC.

Adenoviral particles or empty adenoviral capsids also can be used totransfer nucleic acid delivery vectors by a virus-mediatedcointernalization process as described in U.S. Pat. No. 5,928,944. Thisprocess can be accomplished in the presence of cationic agent(s) such aspolycarbenes or lipid vesicles comprising one or more lipid layers.

Adenoviral particles may be prepared and propagated according to anyconventional technique in the field of the art (e.g., W0 96/17070) usinga complementation cell line or a helper virus, which supplies in transthe missing viral genes necessary for viral replication. The cell lines293 (Graham et al., 1977, J. Gen. Virol. 36: 59-72) and PERC6 (Fallauxet al., 1998, Human Gene Therapy 9: 1909-1917) are commonly used tocomplement E1 deletions. Other cell lines have been engineered tocomplement defective vectors (Yeh, et al., 1996, J. Virol. 70: 559-565;Kroughak and Graham, 1995, Human Gene Ther. 6: 1575-1586; Wang, et al.,1995, Gene Ther. 2: 775-783; Lusky, et al., 1998, J. Virol. 72:2022-203; EP 919627 and W0 97/04119). The adenoviral particles can berecovered from the culture supernatant but also from the cells afterlysis and optionally further purified according to standard techniques(e.g., chromatography, ultracentrifugation, as described in W0 96/27677,W0 98/00524 W0 98/26048 and WO 00/50573).

Cell-type specific targeting may be achieved with vectors derived fromadenoviruses having a broad host range by the modification of viralsurface proteins. For example, the specificity of infection ofadenoviruses is determined by the attachment to cellular receptorspresent at the surface of permissive cells. In this regard, the fiberand penton present at the surface of the adenoviral capsid play acritical role in cellular attachment (Defer, et al., 1990, J. Virol. 64:3661-3673). Thus, cell targeting of adenoviruses can be carried out bygenetic modification of the viral gene encoding fiber and/or penton, togenerate modified fiber and/or penton capable of specific interactionwith unique cell surface receptors. Examples of such modifications aredescribed in Wickarn, et al., 1997, J. Virol. 71: 8221-8229; Arriberg,et al., 1997, Virol. Chem. 268: 6866-6869; Roux, et al., 1989, Proc.Natl. Acad Sci. USA 86: 9079-9083; Miller and Vile, 1995, FASEB J. 9:190-199; W0 93/09221, and in W0 95/28494.

In a particularly preferred aspect, adeno-associated viral sequences areused as vectors. Vectors derived from the human parvovirus AAV-2(adeno-associated virus type 2) are among the most promising genedelivery vehicles currently being developed. Several of the features ofthis system for packaging a single-stranded DNA suggest it as a possiblealternative to naked DNA for delivery of genetic vaccines. A primaryattractive feature, in contrast to other viral vectors such as vacciniaor adenovirus, is that AAV vectors do not express any viral genes. Theonly viral DNA sequences included in the vaccine construct are the 145bp inverted terminal repeats (ITR). Thus, as in immunization with nakedDNA, the only gene expressed is that of the antigen, or antigen chimera.Additionally, AAV vectors are known to transduce both dividing andnon-dividing cells, such as human peripheral blood monocyte-deriveddendritic cells, with persistent transgene expression, and with thepossibility of oral and intranasal delivery for generation of mucosalimmunity. Moreover, the amount of DNA required appears to be much lessby several orders of magnitude, with maximum responses at doses of 10¹⁰to 10¹¹ particles or copies of DNA in contrast to naked DNA doses of 50μg or ˜10¹⁵ copies.

In one aspect, AAV vectors are packaged by co-transfection of a suitablecell line (e.g., human 293 cells) with the DNA contained in the AAV ITRchimeric protein encoding constructs and an AAV helper plasmid ACG2containing the AAV coding region (AAV rep and cap genes) without theITRs. The cells are subsequently infected with the adenovirus Ad5.Vectors can be purified from cell lysates using methods known in the art(e.g., such as cesium chloride density gradient ultracentrifugation) andare validated to ensure that they are free of detectablereplication-competent AAV or adenovirus (e.g., by a cytopathic effectbioassay). AAV titer may be determined by quantitative PCR with virusDNA samples prepared after digestion with proteinase K. Preferably,vector titers produced by such a method are approximately 5×10¹² to1×10¹³ DNase resistant particles per ml.

In other aspects, retroviral vectors are used. Retroviruses are a classof integrative viruses which replicate using a virus-encoded reversetranscriptase, to replicate the viral RNA genome into double strandedDNA which is integrated into chromosomal DNA of the infected cells(e.g., target cells). Such vectors include those derived from murineleukemia viruses, especially Moloney (Gilboa, et al., 1988, Adv. Exp.Med. Biol. 241: 29) or Friend's FB29 strains (W0 95/01447). Generally, aretroviral vector is deleted of all or part of the viral genes gag, poland env and retains 5′ and 3′ LTRs and an encapsidation sequence. Theseelements may be modified to increase expression level or stability ofthe retroviral vector. Such modifications include the replacement of theretroviral encapsidation sequence by one of a retrotransposon such asVL30 (see, e.g., U.S. Pat. No. 5,747,323). Preferably, thepolynucleotide of interest is inserted downstream of the encapsidationsequence, preferably in opposite direction relative to the retroviralgenome. Cell specific targeting may be achieved by the conjugation ofantibodies or antibody fragments to the retroviral envelope protein asis know in the art.

Retroviral particles are prepared in the presence of a helper virus orin an appropriate complementation (packaging) cell line which containsintegrated into its genome the retroviral genes for which the retroviralvector is defective (e.g. gag/pol and env). Such cell lines aredescribed in the prior art (Miller and Rosman, 1989, BioTechniques 7:980; Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85: 6460;Markowitz, et al., 1988, Virol. 167: 400). The product of the env geneis responsible for the binding of the viral particle to the viralreceptors present on the surface of the target cell and, thereforedetermines the host range of the retroviral particle. in the context ofthe invention, it is advantageous to use a packaging cell line, such asthe PA317 cells (ATCC CRL 9078) or 293E16 (W097/35996) containing anamphotropic envelope protein, to allow infection of human and otherspecies' target cells. The retroviral particles are preferably recoveredfrom the culture supernatant and may optionally be further purifiedaccording to standard techniques (e.g. chromatography,ultracentrifugation).

Other suitable viruses include poxviruses. The genome of several membersof poxyiridae has been mapped and sequenced. A poxyiral vector may beobtained from any member of the poxyiridae, in particular canarypox,fowlpox and vaccinia virus. Suitable vaccinia viruses include, but arenot limited to, the Copenhagen strain (Goebel, et al., 1990, Virol. 179:247-266; Johnson, et al., 1993, Virol. 196: 381-401), the Wyeth strainand the modified Ankara (MVA) strain (Antoine, et al., 1998, Virol. 244:365-396). The general conditions for constructing a vaccinia virusvector are known in the art (see, e.g., EP 83 286 and EP 206 920; Mayret al., 1975, Infection 3: 6-14; Sutter and Moss, 1992, Proc. Natl.Acad. Sci. USA 89: 10847-10851). Preferably, the polynucleotide ofinterest is inserted within a non-essential locus such as the nOD7coding intergenic regions or any gene for which inactivation or deletiondoes not significantly impair viral growth and replication.

Poxyiral particles are prepared as described in the art (Piccini, etal., 1987, Methods of Enzymology 153: 545-563; U.S. Pat. No. 4,769,330;U.S. Pat. No. 4,772,848; U.S. Pat. No. 4,603,112; U.S. Pat. No.5,100,587 and U.S. Pat. No. 5,179,993). Generally, a donor plasmid isconstructed, amplified by growth in E. coli and isolated by conventionalprocedures. Then, it is introduced into a suitable cell culture (e.g.chicken embryo fibroblasts) together with a poxvirus genome, to produce,by homologous recombination, poxyiral particles. These can be recoveredfrom the culture supernatant or from the cultured cells after a lysisstep (e.g., chemical lysis, freezing/thawing, osmotic shock, sonicationand the like). Consecutive rounds of plaque purification can be used toremove contaminating wild type virus. Viral particles can then bepurified using the techniques known in the art (e.g., chromatographicmethods or ultracentrifugation on cesium chloride or sucrose gradients).

The use of vaccinia as a live virus vaccine in the global campaign toeradicate smallpox made vaccinia an obvious choice for development as alive recombinant vaccine vector. Live recombinant vaccinia virusesexpressing close to 100 different foreign proteins have been reported,and a number of these are effective experimental vaccines (reviewed byMoss and Flexner, 1987). Vaccinia is particularly versatile as anexpression vector because of its large genomic size, capability ofaccepting at least 25,000 base pairs of foreign DNA, and its ability toinfect most eukaryotic cell types, including insect cells (ibid.).Unlike other DNA viruses, poxviruses replicate exclusively in thecytoplasm of infected cells, reducing the possibility of geneticexchange of recombinant viral DNA with the host chromosome. Recombinantvaccinia vectors have been shown to properly process and expressproteins from a variety of sources including man, other mammals,parasites, RNA and DNA viruses, bacteria and bacteriophage.

The virus is capable of infecting most mammals, making it a usefulvector for studying a broad range of human and animal diseases. Theexpression of DNA encoding a foreign protein is controlled by host virusregulatory elements, including upstream promoter sequences and, wherenecessary, RNA processing signals. Insertion of foreign DNA intononessential regions of the vaccinia virus genome has been carried outby homologous recombination (Panicali, et al., Proc. Nat'l. Acad. Sci,USA, 79: 4927, 1982; Mackett, et al., Proc. Nat'l. Acad. Sci. USA, 79:7415, 1982).

Expression of foreign genes within the DNA may occur because oftranscriptional regulatory elements at or near the site of insertion orby more precise genetic engineering. Plasmid vectors that greatlyfacilitate insertion and expression of foreign genes have beenconstructed (Mackett, et al., J. Virol, 49: 857, 1982). These vectorscontain an expression site, composed of a vaccinia transcriptionalpromoter and one or more unique restriction endonuclease sites forinsertion of the foreign coding sequence flanked by DNA from anonessential region of the vaccinia genome. The choice of promoterdetermines both the time (e.g., early or late) and level of expression,whereas the flanking DNA sequence determines the site of homologousrecombination.

Only about one in a thousand virus particles produced by this procedureis a recombinant. Although recombinant virus plaques can be identifiedby DNA hybridization, efficient selection procedures have beendeveloped. By using segments of nonessential vaccinia virus thymidinekinase (TK) gene as flanking sequences, the foreign gene recombines intothe TK locus and by insertion inactivates the TK gene. Selection of TKvirus is achieved by carrying out the virus plaque assay in TK cells inthe presents of 5-bromodeoxyuridine. Phosphorylation of the nucleosideanalogue and consequent lethal incorporation into viral DNA occurs onlyin cells infected with TK.sup.+ parental virus. Depending on theefficiency of the transfection and recombination, up to 80 of theplaques are desired recombinants, and the rest are spontaneous TKmutants.

Plasmid vectors that contain the E. coliβ-galactosidase gene, as well asan expression site for a second gene, permit an alternative method ofdistinguishing recombinant from parental virus (Chakrabarti, et al.,Mol. Cell. Biol., 5: 3403, 1985). Plaques formed by such recombinantscan be positively identified by the blue color that forms upon additionof an appropriate indicator. By combining both TK selection andbeta-galactosidase expression, recombinant virus is readily and quicklyisolated. The recombinants are then amplified by propagation in suitablecell lines and expression of the inserted gene is checked by appropriateenzymological, immunological or physical procedures.

An upper limit to the amount of genetic information that can be added tothe vaccinia virus genome is not yet known. However, the addition ofnearly 25,000 base pairs of foreign DNA had no apparent deleteriouseffect on virus yield (Smith, et al., Gene, 25:21, 1983). Were itnecessary, large segments of the vaccinia virus genome could be deletedto provide additional capacity (Moss, et al., J. Virol. 40: 387, 1981).

Viral capsid molecules may include targeting moieties to facilitatetargeting and/or entry into cells. Suitable targeting molecules,include, but are not limited to: chemical conjugates, lipids,glycolipids, hormones, sugars, polymers (e.g. PEG, polylysine, PEI andthe like), peptides, polypeptides (see, e.g., WO 94/40958), vitamins,antigens, lectins, antibodies and fragments thereof. Preferably, suchtargeting molecules recognize and bind to cell-specific markers,tissue-specific markers, cellular receptors, viral antigens, antigenicepitopes or tumor-associated markers.

A composition based on viral particles may be formulated in the form ofdoses of between 10 and 10¹⁴ i.u. (infectious units), and preferably,between 10 and 10¹¹ i.u. The titer may be determined by conventionaltechniques. The doses of nucleic acid delivery vector are preferablycomprised between 0.01 and 10 mg/kg, more especially between 0.1 and 2mg/kg.

Cell-Based Delivery Vehicles

The nucleic acid vectors according to the invention can be delivered totarget cells by means of other cells (“delivery cells) which comprisethe vectors. Methods for introducing vectors into cells are known in theart and include microinjection of DNA into the nucleus of a cell(Capechi, et al., 1980, Cell 22: 479-488); transfection with CaP0₄ (Chenand Okayama, 1987, Mol. Cell. Biol. 7: 2745 2752), electroporation (Chu,et al., 1987, Nucleic Acid Res. 15: 1311-1326); lipofection/liposomefusion (Felgner, et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7413-7417)and particle bombardment (Yang, et al., 1990, Proc. Natl. Acad. Sci. USA87: 9568-9572). Suitable cells include autologous and non-autologouscells, and may include xenogenic cells. Delivery cells may be induced todeliver their contents to the target cells by inducing their death(e.g., by providing inducible suicide genes to these cells).

Accessory Molecules

The compositions according to the invention may comprise one or moreaccessory molecules for facilitating the introduction of a nucleic aciddelivery vector into a cell and/or for enhancing a particulartherapeutic effect.

In addition, the composition according to the present invention mayinclude one or more stabilizing substance(s), such as lipids, nucleaseinhibitors, hydrogels, hyaluronidase (W0 98/53853), collagenase,polymers, chelating agents (EP 890362), in order to inhibit degradationwithin the animal/human body and/or improve transfection/infection ofthe vector into a target cell. Such substances may be used alone or incombination (e.g., cationic and neutral lipids).

It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Themixture of adenoviruses to solutions containing a lipid-complexed DNAvector or the binding of DNA to polylysine covalently attached toadenoviruses using protein cross-linking agents may substantiallyimprove the uptake and expression of a nucleic acid delivery vector(see, e.g., Curiel, et al., 1992, Am. I. Respir. Cell. Mol. Biol. 6:247-252).

Host Cells

Nucleic acid vectors according to the invention can be expressed in avariety of host cell, including, but not limited to: prokaryotic cells(e.g., E. coli, Staphylococcus sp., Bacillus sp.); yeast cells (e.g.,Saccharomyces sp.); insect cells; nematode cells; plant cells; amphibiancells (e.g., Xenopus); avian cells; and mammalian cells (e.g., humancells, mouse cells, mammalian cell lines, primary cultured mammaliancells, such as from dissected tissues).

The molecules can be expressed in host cells isolated from an organism,host cells which are part of an organism, or host cells which areintroduced into an organism. In one aspect, fusion molecules areexpressed in host cells in vitro, e.g., in culture. In another aspect,fusion molecules are expressed in a transgenic organism (e.g., atransgenic mouse, rat, rabbit, pig, primate, etc.) that comprisessomatic and/or germline cells comprising nucleic acids encoding thefusion molecules. Methods for constructing transgenic animals are wellknown in the art and are routine.

Nucleic acid vectors also can be introduced into cells in vitro, and thecells (e.g., such as stem cells, hematopoietic cells, lymphocytes, andthe like) can be introduced into the host organism. The cells may beheterologous or autologous with respect to the host organism. Forexample, cells can be obtained from the host organism, nucleic acidvectors introduced into the cells in vitro, and then reintroduced intothe host organism.

Antigen Presenting Cells

In a preferred aspect of the invention, a nucleic acid delivery vehiclesuch as described above is introduced into a natural or engineeredantigen presenting cell.

The term “antigen presenting cell” (APC) as used herein intends any cellwhich presents on its surface an antigen in association with a majorhistocompatibility complex molecule, preferably a class II molecule, orportion thereof. Examples of suitable APCs are discussed in detail belowand include, but are not limited to, whole cells such as macrophages,dendritic cells, B cells, hybrid APCs, and foster antigen presentingcells. Methods of making hybrid APCs are described and known in the art.

Dendritic cells (DCs) are potent antigen-presenting cells. It has beenshown that DCs provide all the signals required for T cell activationand proliferation. These signals can be categorized into two types. Thefirst type, which gives specificity to the immune response, is mediatedthrough interaction between the T-cell receptor/CD3 (“TCR/CD3”) complexand an antigenic peptide presented by a major histocompatibility complex(“MHC” defined above) class I or II protein on the surface of APCs. Thisinteraction is necessary, but not sufficient, for T cell activation tooccur. In fact, without the second type of signals, the first type ofsignals can result in T cell anergy. The second type of signals, calledco-stimulatory signals, is neither antigen-specific nor MHC-restricted,and can lead to a full proliferation response of T cells and inductionof T cell effector functions in the presence of the first type ofsignals.

Several molecules have been shown to enhance co-stimulatory activity.These include, but are not limited to, heat stable antigen (HSA),chondroitin sulfate-modified MHC invariant chain (Ii-CS), intracellularadhesion molecule I (ICAM-1), and B7 co-stimulatory molecule on thesurface of APCs and its counter-receptor CD28 or CTLA-4 on T cells.

Other important co-stimulatory molecules are CD40, CD54, CD80, CD86. Asused herein, the term “co-stimulatory molecule” encompasses any singlemolecule or combination of molecules which, when acting together with apeptide/MHC complex bound by a TCR on the surface of a T cell, providesa co-stimulatory effect which achieves activation of the T cell thatbinds the peptide. The term thus encompasses B7, or other co-stimulatorymolecule(s) on an APC, fragments thereof (alone, complexed with anothermolecule(s), or as part of a fusion protein) which, together withpeptide/MHC complex, binds to a cognate ligand and result in activationof the T cell when the TCR on the surface of the T cell specificallybinds the peptide. Co-stimulatory molecules are commercially availablefrom a variety of sources, including, for example, Beckman Coulter.

In one aspect of the invention, the method described in Romani et al.,J. Immunol. Methods 196: 135-151, 1996, and Bender et al, J. Immunol.Methods 196: 121-135, 1996, are used to generate both immature andmature dendritic cells from the peripheral blood mononuclear cells(PBMCs) of a mammal, such as a murine, simian or human. Briefly,isolated PBMCs are pre-treated to deplete T- and B-cells by means of animmunomagnetic technique. Lymphocyte-depleted PBMC are then cultured forin RPMI medium 9 e.g., about 7 days), supplemented with human plasma(preferably autologous plasma) and GM-CSF/IL-4, to generate dendriticcells. Dendritic cells are nonadherent when compared to their monocyteprogenitors. Thus, on approximately day 7, non-adherent cells areharvested for further processing.

The dendritic cells derived from PBMC in the presence of GM-CSF and IL-4are immature, in that they can lost the nonadherence property and revertback to macrophage cell fate if the cytokine stimuli are removed fromthe culture. The dendritic cells in an immature state are very effectivein processing native protein antigens for the MHC class II restrictedpathway (Romani, et al., J. Exp. Med. 169:1169, 1989). Furthermaturation of cultured dendritic cells is accomplished by culturing for3 days in a macrophage-conditioned medium (CM), which contains thenecessary maturation factors. Mature dendritic cells are less able tocapture new proteins for presentation but are much better at stimulatingresting T cells (both CD4 and CD8) to grow and differentiate.

Mature dendritic cells can be identified by their change in morphology,such as the formation of more motile cytoplasmic processes; by theirnonadherence; by the presence of at least one of the following markers:CD83, CD68, HLA-DR or CD86; or by the loss of Fc receptors such as CD115 (reviewed in Steinman, Annu. Rev. Immunol. 9: 271, 1991). Maturedendritic cells can be collected and analyzed using typicalcytofluorography and cell sorting techniques and devices, such asFACScan and FACStar. Primary antibodies used for flow cytometry arethose specific to cell surface antigens of mature dendritic cells andare commercially available. Secondary antibodies can be biotinylated Igsfollowed by FITC- or PE-conjugated streptavidin.

Alternatively, others have reported that a method for upregulating(activating) dendritic cells and converting monocytes to an activateddendritic cell phenotype. This method involves the addition of calciumionophore to the culture media convert monocytes into activateddendritic cells. Adding the calcium 21 ionophore A23187, for example, atthe beginning of a 24-48 hour culture period resulted in uniformactivation and dendritic cell phenotypic conversion of the pooled“monocyte plus DC” fractions: characteristically, the activatedpopulation becomes uniformly CD14 (Leu M3) negative, and upregulatesHLA-DR, HLA-DQ, ICAM-1,137.1, and 137.2. Furthermore, this activatedbulk population functions as well on a small numbers basis as a furtherpurified. Specific combination(s) of cytokines have been usedsuccessfully to amplify (or partially substitute) for theactivation/conversion achieved with calcium ionophore: these cytokinesinclude but are not limited to G-CSF, GM-CSF, IL-2, and IL-4. Eachcytokine when given alone is inadequate for optimal upregulation.

The second approach for isolating APCs is to collect the relativelylarge numbers of precommitted APCs already circulating in the blood.Previous techniques for isolating committed APCs from human peripheralblood have involved combinations of physical procedures such asmetrizamide gradients and adherence/nonadherence steps (Freudenthal etal. PNAS 87: 7698-7702, 1990); Percoll gradient separations(Mehta-Damani, et al., J. Immunol. 153: 996-1003, 1994); andfluorescence activated cell sorting techniques (Thomas et al., J.Immunol. 151: 6840-52, 1993).

It should be obvious to those of skill in the art that there are manymethods routine in the art for isolating professional antigen presentingcells (or their precursors) and that such methods and others which maybe developed are not limiting and are encompassed within the scope ofthe invention.

In one embodiment, the APCs and therefore the cells presenting one ormore antigens are autologous. In another embodiment, the APCs presentingthe antigen are allogeneic, i.e., derived from a different subject.

As discussed above, nucleic acids encoding chimeric molecules can beintroduced into APCs using the methods described above or others knownin the art, including, but not limited to, transfection,electroporation, fusion, microinjection, viral-based delivery, or cellbased delivery. Arthur et al., Cancer Gene Therapy 4(1):17-25, 1997,reports a comparison of gene transfer methods in human dendritic cells.

Known, partial and putative human leukocyte antigen (HLA), the geneticdesignation for the human MHC, amino acid and nucleotide sequences,including the consensus sequence, are published (see, e.g., Zemmour andParham, Immunogenetics 33: 310-320, 1991), and cell lines expressing HLAvariants are known and generally available as well, many from theAmerican Type Culture Collection (“ATCC”). Therefore, using PCR, MHCclass II-encoding nucleotide sequences are readily operatively linked toan expression vector of this invention that is then used to transform anappropriate cell for expression therein.

Professional APCs can be used, such as macrophages, B cells, monocytes,dendritic cells, and Langerhans cells. These are collected from theblood or tissue of 1) an autologous donor; 2) a heterologous donorhaving a different HLA specificity then the host to be treated; or 3)from a xenogeneic donor of a different species using standard procedures(Coligan, et. al., Current Protocols in Immunology, sections 3 and 14,1994). The cells may be isolated from a normal host or a patient havingan infectious disease, cancer, autoimmune disease, or allergy.

Professional APCs may be obtained from the peripheral blood usingleukopheresis and “FICOLL/HYPAQUE” density gradient centrifugation(stepwise centrifugation through Ficoll and discontinuous Percolldensity gradients). Procedures are utilized which avoid the exposure ofthe APCs to antigens which could be internalized by the APCs, leading toactivation of T cells not specific for the antigens of interest.

Cells which are not naturally antigen presenting can be engineered to beantigen presenting by introducing sequences encoding appropriatemolecules. For example, nucleic acid sequences encoding MHC class IImolecules, accessory molecules, co-stimulatory molecules and antigenprocessing assisting molecules can be introduced after direct synthesis,cloning, purification of DNA from cells containing such genes, and thelike. One expedient means to obtain genes for encoding the moleculesused in the compositions and methods described herein is by polymerasechain reaction (PCR) amplification on selected nucleic acid templateswith selected oligonucleotide primer pairs. For example, epithelialcells, endothelial cells, tumor cells, fibroblasts, activated T cells,eosinophils, keratinocytes, astrocytes, microglial cells, thymiccortical epithelial cells, Schwann cells, retinal pigment epithelialcells, myoblasts, vascular smooth muscle cells, chondrocytes,enterocytes, thyrocytes and kidney tubule cells can be used. These maybe primary cells recently explanted from a host and not extensivelypassaged in cell culture to form an established cell line, orestablished cell lines that are relatively homogeneous and capable ofproliferating for many generations or indefinitely.

Cells that are not professional APCs are isolated from any tissue of anautologous donor; a heterologous donor or a xenogeneic donor, where theyreside using a variety of known separation methods (Darling, AnimalCells: Culture and Media. J. Wiley, New York, 1994; Freshney, Culture ofAnimal Cells. Alan R. Liss, Inc., New York, 1987). Non-autologous cells,e.g., heterologous or xenogeneic cells, can be engineered ex vivo toexpress HLA class I and class II molecules that match known human HLAspecificities. These cells can then be introduced into a human subjectmatching the HLA specificity of the engineered cells. The cells arefurther engineered ex vivo to express one or more chimeric vaccinesaccording to the invention.

The engineered cells are maintained in cell culture by standard cellculture methods (Darling, Animal Cells: Culture and Media”. J. Wiley,New York, 1994; Freshney, Culture of Animal Cells”. Alan R. Liss, Inc.,New York, 1987). Cell lines for use in the present invention areobtained from a variety of sources (e.g., ATCC Catalogue of Cell Lines &Hybidomas, American Type Culture Collection, 8th edition, 1995), or areproduced using standard methods (Freshney, Culture of ImmortalizedCells, Wiley-Liss, New York, 1996). Non-transformed cell lines arepreferred for use in human subjects.

In one aspect, CD34⁺ precursors that are differentiating under theinfluence of GM-CSF into dendritic cells are obtained from the body of asubject and nucleic acids encoding chimeric vaccines according to theinvention are introduced into the cells, which are then re-injected intothe subject. Utilizing the construct containing antigenic sequenceslinked to an endosomal/lysosomal targeting signal (and preferablycomprising a LAMP-like lumenal polypeptide) will enhance the associationof peptides derived from a particular antigen with MHC class IImolecules on the transduced antigen presenting cells, resulting insignificantly more potent systemic T cell dependent immune responses.While the antigen presenting cells transfected in this strategy arepreferably autologous cells, any MHC class II cells that effectivelypresent antigen in the host may be used as described above.

Peptide Vaccines

Also within the scope of this invention are vaccines containingcell-free peptide immunogens, where the immunogen contains an antigendomain and sequences of a lysosomal membrane polypeptide (e.g., such asa LAMP polypeptide or a homolog, ortholog, variant, or modified versionthereof) or sequences of an endocytic receptor for targeting andtrafficking the antigen to an endosomal/lysosomal compartment orlysosome-related organelle for binding to an MHC class II molecule orfor delivery to another compartment/organelle for binding to an MHCclass II molecule. Preferably, the antigen is processed within thecompartment/organelle (or subsequent compartment/organelle to which itis delivered) to generate an epitope bound to an MHC class II moleculecapable of modulating an immune response.

The chimeric vaccine may also comprise a transmembrane region and/orcytoplasmic tail with lysosomal targeting region (preferably from a LAMPpolypeptide), and/or di-leucine domain, Tyr motif, MR6 domain, prolinerich domain, and/or Ser-Val-Val domain. The chimeric vaccine also may bebound in a membranous structure to facilitate its administration to thebody of an organism. For example, the chimeric vaccine may beincorporated into liposomes, as described in U.S. Pat. No. 4,448,765.

When a protein or polypeptide is to be used as an immunogen, it may beproduced by expression of any one or more of the DNA constructsdescribed above in a recombinant cell or it may be prepared by chemicalsynthesis. For example, the Merrifield technique (Journal of AmericanChemical Society, vol. 85, pp. 2149-2154, 1968), can be used.

Administration

Vaccine material according to this invention may contain the immunestimulatory constructs described above or may be recombinantmicroorganisms, or antigen presenting cells which express the immunestimulatory constructs. Preparation of compositions containing vaccinematerial according to this invention and administration of suchcompositions for immunization of individuals are accomplished accordingto principles of immunization that are well known to those skilled inthe art.

Large quantities of these materials may be obtained by culturingrecombinant or transformed cells containing replicons that express thechimeric DNA described above. Culturing methods are well-known to thoseskilled in the art and are taught in one or more of the documents citedabove. The vaccine material is generally produced by culture ofrecombinant or transformed cells and formulated in a pharmacologicallyacceptable solution or suspension, which is usually aphysiologically-compatible aqueous solution, or in coated tablets,tablets, capsules, suppositories or ampules, as described in the art,for example in U.S. Pat. No. 4,446,128, incorporated herein byreference. Administration may be any suitable route, including oral,rectal, intranasal or by injection where injection may be, for example,transdermal, subcutaneous, intramuscular or intravenous.

The vaccine composition is administered to a mammal in an amountsufficient to induce an immune response in the mammal. A minimumpreferred amount for administration is the amount required to elicitantibody formation to a concentration at least 4 times that whichexisted prior to administration. A typical initial dose foradministration would be 10-5000 micrograms when administeredintravenously, intramuscularly or subcutaneously, or 10⁵ to 10¹¹ plaqueforming units of a recombinant vector, although this amount may beadjusted by a clinician doing the administration as commonly occurs inthe administration of vaccines and other agents which induce immuneresponses. A single administration may usually be sufficient to induceimmunity, but multiple administrations may be carried out to assure orboost the response.

Vaccines may be tested initially in a non-human mammal (e.g., a mouse orprimate). For example, assays of the immune responses of inoculated micecan be used to demonstrate greater antibody, T cell proliferation, andcytotoxic T cell responses to the lysosome-targeted chimeric proteinsthan to wild type antigen. Chimeric proteins can be evaluated in Rhesusmonkeys to determine whether a DNA vaccine formulation that is highlyeffective in mice will also elicit an appropriate monkey immuneresponse. In one aspect, each monkey receives a total of 5 mg DNA perimmunization, delivered IM and divided between 2 sites, withimmunizations at day 0 and at weeks 4, 8, and 20, with an additionaldoses optional. Antibody responses, ADCC, CD4⁺ and CD8⁺ T-cell cytokineproduction, CD4⁺ and CD8⁺ T-cell antigen-specific cytokine staining canbe measured to monitor immune responses to the vaccine.

Further description of suitable methods of formulation andadministration according to this invention may be found in U.S. Pat. No.4,454,116 (constructs), U.S. Pat. No. 4,681,762 (recombinant bacteria),and U.S. Pat. Nos. 4,592,002 and 4,920,209 (recombinant viruses).

Immune Tolerance and Autoimmunity

Many auto-immune diseases show a correlation with certain MHC class IIhaplotypes and are associated with aberrant auto-antibody production,suggesting that the generation of self-reactive MHC class II restrictedCD4⁺ T cells is an important pathogenetic step. Given that CD4⁺ cellscan, under certain circumstances, be inactivated or anergized byengagement of their T cell receptor in the absence of a second signal(such as the co-engagement of CD28 by its ligand B7), it follows thatthe efficient presentation of an MHC class II restricted antigen on anMHC class II cell that did not display the appropriate second signalwould represent an effective toleragen. The generation of this toleranceor inactivation of certain CD4⁺ T cells could be used to turn offaberrant immune responses in auto-immune diseases.

In the embodiment of this principle, a poor antigen presenting cell(that did not express any co-stimulatory signals) would either beinduced to express MHC class II or would be transfected with theappropriate MHC class II genes. This cell would then be additionallytransduced with the auto-antigen of interest, such as the acetylcholinereceptor in the case of myaesthenia gravis, linked to theendosomal/lysosomal targeting signal. Injection of these cells into thehost would result in turning off T cell responses against the antigen,based on the efficient presentation of peptide sequences on MHC class IImolecules to T cell receptors on CD4⁺ T cells in the absence of theappropriate co-stimulatory signals (signals that are provided byeffective antigen present cells).

Cancer Immunotherapy

Candidates for Prevention and Treatment

Candidates for cancer immunotherapy would be any patient with a cancerpossessing a defined and identified tumor specific antigen whose genecan be cloned and modified by the LAMP lysosomal/endosomal targetingsequences as described herein. Examples include patients with documentedEpstein-Ban virus associated lymphomas, patients with HPV associatedcervical carcinomas, patients with chronic HCV, or patients with adefined re-arrangement or mutation in an oncogene or tumor suppressorgene. It is envisioned that therapy with a vaccine incorporating thetumor antigen linked to the endosomal/lysosomal trafficking andtargeting sequences in a viral vaccine could be utilized at any periodduring the course of the individual's cancer, once it is identified. Itis also possible that in high risk patients, vaccination in order toprevent the subsequent emergence of a cancer with a defined tumorspecific antigen could be performed.

Procedure for Therapy

In one embodiment, recombinant viral vaccine containing the antigenlinked with the lysosomal/endosomal trafficking and targeting sequenceincorporated into a viral vaccine such as vaccinia, would be produced inlarge quantities as described above and would be injected into thepatient at any suitable time during the course of their malignancy.Preferably, the vaccine would be injected at a stage when the tumorburden was low. In an alternative embodiment in which this construct isintroduced into the individual's antigen presenting cells, precursors tothe antigen presenting cells or mature antigen presenting cells aredrawn either from the individual's bone marrow or peripheral blood byvena puncture. These cells are established in culture followed bytransduction with the chimeric construct. Once transduction hadoccurred, these antigen presenting cells are injected back into thepatient.

In a particularly preferred embodiment, the invention provides a methodof treatment for a cancer patient having low tumor burden, such as earlyin the disease, after resection of a neoplastic tumor, or when theburden of tumor cells is otherwise reduced. In this method, once atumor-specific cell surface antigen characteristic of the patient'stumor has been identified, a cell population containing autologous stemcells capable of differentiation into antigen presenting cells whichwill express MHC class II molecules is obtained from the patient. Thesecells are cultured and transformed by introducing a heterologous orchimeric DNA molecule which encodes a protein containing at least oneepitope of the tumor-specific antigen found on the cells of thepatient's tumor and a lumenal trafficking domain of a lysosomeassociated membrane polypeptide (e.g., LAMP polypeptide, homolog,ortholog, variant, or modified form thereof) or the lumenal traffickingdomain of an endocytic receptor, and a cytoplasmic targeting domain of aLAMP polypeptide, homolog, ortholog, variant, or modified form thereof,or of an endocytic receptor for targeting the antigen to anendosomal/lysosomal compartment or lysosome-related organelle and forassociation with an MHC class II molecule either within thecompartment/organelle or within another compartment/organelle to whichthe antigen is delivered. Such chimeric DNA molecules can encodeadditional domain sequences as described above (e.g., sequences encodingtransmembrane domains; signal sequences, cytoplasmic domains fortargeting to an endosomal/lysosomal compartment or lysosome-relatedorganelles, di-leucine domains, Tyr motif domains, proline rich domains,Ser-Val-Val domains, and the like).

The transfected stem cell population is then reintroduced into thepatient, where the stem cells differentiate into antigen presentingcells which express MHC class II molecules complexed with T_(h) epitopesfrom the tumor-specific antigen. The immune response to thetumor-specific antigen will be enhanced by enhanced stimulation of thehelper T cell population.

More generally, in one embodiment, this invention provides a vaccinecomposition for modulating an immune response in a mammal to an antigen(i.e., stimulating, enhancing, or reducing such a response). Preferably,the composition comprises a vaccine vector, wherein the vector containsa chimeric DNA segment which encodes a protein containing at least oneepitope of the tumor-specific antigen found on the cells of thepatient's tumor and a lumenal trafficking domain of a lysosomeassociated membrane polypeptide (e.g., LAMP polypeptide, homolog,ortholog, variant, or modified form thereof) or the lumenal traffickingdomain of an endocytic receptor, and a targeting domain, such as thecytoplasmic targeting domain of a LAMP polypeptide, homolog, ortholog,variant, or modified form thereof, or of an endocytic receptor, fortargeting the antigen to an endosomal/lysosomal compartment orlysosome-related organelle and for association with an MHC class IImolecule either within the compartment/organelle or within anothercompartment/organelle to which the antigen is delivered. Such chimericDNA molecules can encode additional domain sequences as described above(e.g., sequences encoding transmembrane domains; signal sequences,cytoplasmic domains for targeting to an endosomal/lysosomal compartmentor lysosome-related organelles, di-leucine domains, Tyr motif domains,proline rich domains, Ser-Val-Val domains, and the like).

Kits

The invention further comprises kits to facilitate performing themethods described herein. In one aspect, a kit comprises a nucleic acidvector as described above and a cell for receiving the vector. The kitmay additionally comprise one or more nucleic acids for engineering thecell into a professional APC. In one aspect, however, the cell is aprofessional APC. The cell may or may not express co-stimulatorymolecules. In a preferred aspect, when the cell does not expressco-stimulatory molecules, the antigen encoded by the vector is anautoantigen. In another aspect, a panel of cells is provided expressingdifferent MHC molecules (e.g., known to be expressed in human beings).In a further aspect, the kit comprises reagents to facilitate entry ofthe vectors into a cell (e.g., lipid-based formulations, viral packagingmaterials, cells, and the like). In still a further aspect, one or moreT cell lines specific for the antigen encoded by the vector is provided,to verify the ability of the vector to elicit, modulate, or enhance animmune response.

EXAMPLES

The invention will now be further illustrated with reference to thefollowing examples. It will be appreciated that what follows is by wayof example only and that modifications to detail may be made while stillfalling within the scope of the invention.

Example 1 High Level Expression HIV-1 Gag In Vitro by PlasmidsContaining Antigen Inserted into the Full LAMP Sequences and the AdenoAssociated Virus Inverted Terminal Repeat Sequences

A number of different nucleic acid constructs were constructed encodingchimeric vaccines.

A plasmid capable of eliciting a high level of Gag expression as afusion protein with LAMP in transfected cells is shown in FIG. 1. Gagexpression by this plasmid is compared to that of a number of otherplasmid constructs in the figure. For example, Plasmid 1 comprises thewild type [HIV-1] Gag gene in the pcDNA3.1 (Invitrogen) vector backbone.Plasmid 2 comprises a gag chimera containing LAMP signal sequence,transmembrane and cytoplasmic domains, in pcDNA3.1. Plasmid 3 comprisesa chimera with Gag inserted between the lumenal andtransmembrane/cytoplasmic domains of the complete LAMP cDNA in pcDNA3.1.Plasmid 4 shows HIVGagΔINS¹⁵ containing mutations of the Gag inhibitorysequences in pcDNA3.1. Plasmid 5 comprises HIVGagΔINS¹⁵ with the LAMPsignal sequence, transmembrane and cytoplasmic domains pcDNA3.1. Plasmid6 comprises p43 vector backbone containing the AAV-ITRs and encodingwild type Gag. Plasmid 7 shows a p43 vector backbone containing gaginserted into the full length LAMP as in plasmid 3.

The Data show, as expected, that the control plasmid (#1) and the onecontaining the Gag/LAMP transmembrane and cytoplasmic tail chimera (#2)did not show significant protein expression. In contrast, thisexperiment resulted in the novel finding of strong Gag expression withthe gag gene included within the full LAMP gene (#3). The amount of Gagprotein expressed was notably greater than that of the plasmid encodingGag with the mutated INS sequences alone (#4) or as a chimera with theLAMP transmembrane and cytoplasmic tail (#5). A further, and even moreremarkable finding was the relatively great Gag protein expression ofthe p43 plasmid containing the AAV ITR sequences and the gag geneincorporated in the complete LAMP sequence (#7). The same p43 plasmidconstruct but without the LAMP sequences showed very little Gagexpression (#6). Similar protein expression as observed in with plasmid#7 in this experiment was observed after transfection of several celllines (COS, 293T, 3T3).

Thus, inserting HIV Gag near the extracellular side of the transmembranedomain of the full-length mouse LAMP-1 results in a much greaterexpression of HIV Gag as a fusion protein with LAMP as compared to thewild type Gag or Gag modified only with the LAMP endoplasmic reticulumtranslocation signal sequence, and Gag expression that was greater eventhan that of the plasmid encoding Gag with the mutated INS sequences. Inaddition, Gag expression of our new HIV Gag-LAMP chimera was remarkablyfurther enhanced by a plasmid DNA vector containing non-translatedsequences of the inverted terminal repeats (ITRs) of theadeno-associated virus (AAV) (p43 vector).

Example 2 Evaluation of the Immune Response of Mice to Plasmids Encodinga Complete LAMP/HIV-1 Gag Chimera Protein

High level expression in transfected cells of Gag protein as a chimeraof approximately 200 kDa (assayed by anti-Gag antibodies) was found toresult from insertion of the HIV-1 p55Gag sequence downstream of thecoding sequence of the LAMP lumenal domain (1113 bp) (FIG. 7E construct#3). Moreover Gag expression was further markedly amplified by includingAAV ITRs into the expression vector. Approximately 40-fold greaterexpression was observed for this construct than for a LAMP/Gag constructwithout such sequences as determined by densitometry. This expressionwas 200-fold greater than the expression of wild type Gag (FIG. 7Econstruct #7). There was minimal Gag protein expression in transfectedcells with plasmids that lacked the LAMP lumenal domain, even inplasmids containing the ITR (FIG. 7E, constructs #1, 2, 6). Thus, thehigh level expression of Gag required the lumenal domain of LAMP.

The cellular localization of LAMP/Gag was analyzed by transfecting themouse fibroblast cell line DCEK.ICAM.Hi7, which expresses MHC class III-Ek molecules, ICAM-1, and B7-1, and can present pre-processed peptidesto naïve CD4 T cells. Class II molecules of this cell line have beenshown to colocalize with LAMP-1. Monoclonal anti-Gag staining of DCEKcells transfected with wild type Gag revealed a diffuse cytoplasmicdistribution of the wild type protein. In contrast, DCEK transfectedwith the LAMP/Gag construct and stained with anti-Gag showed a strikingcolocalization of the LAMP/Gag chimera with MHC II in several highlylabeled vesicles.

The ability of the chimeric proteins to modulate an immune response wasalso evaluated. Groups of six mice were immunized with 50 μg of DNA atthe base of the tail on days 0, 30, 60 and 90 with the following plasmidconstructs as follows. Blood samples were obtained on days—1, 29, 59,and 89, and half of the mice sacrificed on days 70 and 100. The effectsof various constructs were examined: 1) pcDNA wild type Gag; 2) pcDNALAMP signal sequence/Gag/LAMP transmembrane and cytoplasmic domains; 3)pcDNA lumenal domain of LAMP/Gag/LAMP transmembrane and cytoplasmicdomains; 4) pcDNA Gag mutated inhibitory sequences; 5) pcDNA signalsequence/mutated inhibitory sequences/LAMP transmembrane and cytoplasmicdomains; 6) p43 (AAV-ITR) wild type Gag; and 7) p43 (AAV-ITR) lumenaldomain of LAMP/Gag/LAMP transmembrane and cytoplasmic domains.

The HIV specific antibody response in each mouse was assayed at 1:100dilution of the plasma collected on day 59, after two immunizations. Thedata are shown as the average of these individual assays (n=5) (FIG. 2).The Gag gene encoded in the full length LAMP cDNA, particularly thatcontained in the p43 vector, elicited a much stronger immune responsethan did any of the other constructs. The GagINS constructs elicitedlesser responses whereas there was no response to any of the wild typeGag gene constructs. The combination of the full-length LAMP/gag chimeraand the AAV plasmid are remarkably more immunogenic than any of theconventional DNA vaccine constructs.

The pooled sera collected after the second to fourth immunizations, days59, 89, and 119, were also tested for the titer of the antibody response(FIG. 3). The Gag-specific antibody titer of mice immunized with Gagsequences combined with the complete LAMP sequence in the p43 vectorwere markedly greater than those of any of the other immunogens, with atiter of ˜300,000 in repeated experiments. Moreover, the titer of theanti-Gag response was maximal at two immunizations. The pcDNA3.I plasmidcontaining Gag sequences in the complete LAMP sequence also elicited asignificant, but much lower titer. The remaining plasmids, including thepcDNA3.1 GagΔINS, showed little immunogenicity in this experiment.

Additional experiments with the secreted LAMP/Gag construct lacking thetransmembrane and cytoplasmic domains, and a plasma membrane LAMP/Gagconstruct lacking the YQTI lysosomal targeting sequence demonstratedthat the enhanced antibody response of the LAMP/Gag DNA requiredlysosomal trafficking. Thus, high protein expression of a secreted Gagfailed to elicit a response comparable to the LAMP trafficked Gag,despite the expectation that a secreted Gag would favor an antibodyresponse. Additionally, here was no greater antibody response byco-injecting DNA encoding LAMP/Gag and Gag wild type proteins, implyingthat additional Gag protein not trafficked to lysosomes had no effect inthe antibody response and that the limiting element in this immunizationwas the CD4⁺ T-helper response.

Assays of CD4-mediated IL-2 and IL-4 mRNA expression, and IFN-7 proteinproduction, upon stimulation of spleen cells with the p55 Gagrecombinant protein all show a much stronger response to immunizationwith the LAMP/Gag plasmid than with plasmids lacking the complete LAMP(FIGS. 7A-D). While this difference could be attributed to the lack ofGag protein expression in the case of immunization with Gag wild typeDNA, the LAMP/secreted Gag was also inferior as an immunogen. Theresults were specific to CD4⁺ cells as IFN-7 production was notinhibited by prior incubation of splenocytes with an anti-CD8 monoclonalantibody, whereas it was completely blocked by treatment of the cells byan anti-CD4 antibody. Combined immunization with Gag wild type andLAMP/Gag did not lead to an increased CD4 response.

DNA vaccines are able to generate CD8⁺ T-cell responses but maximalpriming of naive CD8⁺ cells requires CD4⁺ activity. Gag-specificCD8⁺-mediated responses to a single immunization of 50 μg DNA weremeasured by in vivo expansion with recombinant vaccinia-Gag-Pol(rVVGag-Pol) for 5 days and activation by the immuno-dominant H2Kd-restricted Gag peptide for 2 hrs. Mice immunized with LAMP/gag DNAuniformly showed a markedly greater CD8⁺ response than did thoseimmunized with wild type gag or the secreted LAMP/Gag chimera. Theresults were comparable with each of three assays, Gag tetramer binding,intra-cellular IFN-γ staining, and Gag-specific cell killing. Removal ofCD8⁺ cells from the effector population abolished the effect.Co-immunization with a plasmid encoding wild type Gag in order toincrease Gag protein delivered to the MHC I pathway of transfected cellsdid not enhance the CD8⁺-mediated responses, indicating that despiteLAMP targeting of antigen there was sufficient MHC I presentation ofantigen.

Dose-response to immunization with pITR/LAMP/Gag. Mice twice immunizedi.m. with 0.1 to 50 mg of LAMP/gag DNA showed a comparable response to10 and 50 mg in assays of Gag-specific antibody response, CD4⁺ INF-7production, and the % INF-γ⁺, CD8⁺ T cells. Cellular responses appearedto require smaller amounts of DNA, with maximum CD4⁺ and CD8⁺ responseswith 10 mg DNA and a significant CD8⁺ response with only 1 mg. Othershave reported that higher doses of immunogen are necessary to inducehumoral immunity as compared to cellular responses.

Example 3 Further Evaluation of the Immune Response of Mice to HIV-1 GagEncoded as a Full Length LAMP Chimera in the p43 Plasmid Vector; aRepeat Study and Additional Controls

Groups of 6 mice were immunized with 50 μg of DNA at the base of thetail on days 0 and 30 with the plasmid constructs as below. Bloodsamples were obtained on days—1, 29, and 45, and half of the micesacrificed on day 45. The primary goal of this experiment, was toconfirm the initial result of the p43(AAV-ITR) LAMP/Gag/TMCD plasmid andto include additional control immunizations. A number of differentconstructs were evaluated:

1) p43 (AAV-ITR) (plasmid negative control); 2) p43 (AAV-ITR) LAMP (LAMPnegative control without gag); 3) p43 (AAV-ITR) wild type Gag; 4) p43(AAV-ITR) lumenal domain of LAMP/Gag/LAMP transmembrane and cytoplasmicdomains; 5) pVax wild type Gag; and 6) pVax lumenal domain ofLAMP/Gag/LAMP transmembrane and cytoplasmic domains.

The anti-HIV specific antibody response at day 29 after the firstimmunization and 15 days after the second immunization; is given as theaverage of the results from individual mice at a 1.300 dilution of theplasma sample (n=6) (FIG. 4A). The data again show that the predominantresponse was after the second immunization of mice innoculated with thep43 (AAV-ITR) lumenal domain of LAMP/Gag/LAMP transmembrand andcytoplasmic domains. This positive response can be attributed to twofactors: the increased protein expression of Gag by the p43 plasmid withthe Gag gene inserted into the LAMP sequences, and the LAMP traffickingof the protein.

The titer of total IgG. IgG1 and IgG2a were determined. The miceinnoculated with the p43 (AAV-ITR) lumenal domain of LAMP/Gag/LAMPtransmembrand and cytoplasmic domains presented a titer of total IgG andIgG1 of 1:24,300 whereas the Gag WT presented a titer of 1:900 (FIG. 6,A). In addition, the p43 (AAV-ITR) lumenal domain of LAMP/Gag/LAMPtransmembrane and cytoplasmic domains presented a titer of IgG2a of1:300 while the Gag WT did not show any IgG2a.

The Gag specific T cell interleukin-4 and INF gamma response of miceimmunized as described in FIG. 5, was measured at day 45 by real timePCR assay (FIGS. 7 and 8). Spleen cells were stimulated overnight bymedium (control), and medium containing 5 μg Gag protein. A strongGag-specific response of cells from the p43 LAMP/Gag plasmid immunizedmice was observed, indicative of the stimulation of Th2 cells. Together,the T cell production of both INFγ and IL-4 suggests a balanced T cellresponse to the immunization.

Example 4 Cell C-Type Lectin Receptor/Gag DNA Chimera Targeting to MHCII (LAMP/Gag/DCLR)

Dendritic cell C-type lectin receptors (DCLR) internalize bound antigensby adsorptive endocytosis and traffic to the MHC II compartment wherethey co-localized with both the multicellular and DC-LAMP molecules. TheDCLR may also act in controlling the character of the immune response.Studies of the DEC-205 (DEC) transmembrane and cytoplasmic sequences assignals for the trafficking of a chimera LAMP/p55Gag/DCLR protein wereperformed. The LAMP lumenal domain sequences are retained in thisconstruct in order to enhance Gag protein expression. The preliminarydata show that cells transfected with the LAMP/Gag/DCLR constructexpress high levels of the Gag protein, that Gag is colocalized withLAMP and MHC II in the transfected cells, and that immunized mice elicitan effective antibody and strong CD4⁺ responses, but possibly limitedCD8⁺ T cell responses.

Plasmid Constructs

RNA from C57BL/6 mouse bone marrow cells was used as template forcloning the transmembrane domain and cytoplasmic 31 amino acid tail ofDEC205 as a model DCLR system. These DEC domains were then used toreplace the corresponding LAMP transmembrane fragment and cytosolic 11amino acid tail of the LAMP/Gag construct in the pITR vector backbone.The membrane-proximal coated pit and distal EDE sequences of DEC205 arebelieved to play roles in DEC205 recycling between plasma membrane andlate endosome/lysosome compartment (Mahnke, et al, 2000).

The following combinations of this sequence were synthesized in theAAV-ITR-containing vector backbone (pITR) (FIG. 8): mLAMP/DEC (negativecontrol, without antigen); p55Gag (wild type HIV-1 Gag); mLAMP/p55Gag(multicellular mLAMP/p55Gag construct as a positive control);mLAMP/p55Gag/DEC (experimental construct); LAMP/p55Gag/DECΔ₇ (controlconstruct terminated at amino acid 7 of the cytoplasmic domain of DEC,thus lacking the MHC II targeting sequences).

LAMP/Gag/DCLR Chimera Protein Expression

Expression of Gag was analyzed with transfected COS-7 cells (FIG. 9). Asexpected, there was detectable wild type Gag expressed by the pITR/gagplasmid (lane 2), and greatly enhanced levels of the LAMP/Gag/DCLR (lane3 & 4) and LAMP/Gag (lane 5) protein chimeras. This over exposed gelalso reveals many degraded fragments of the LAMP/Gag chimeras reactingwith the anti-Gag antibody.

Cellular Trafficking of Protein Chimeras Containing the DEC DCLRCytoplasmic Domain.

The cellular localization of LAMP/DCLR and LAMP/Gag/DCLR chimeras intransfected human 293 cells have been examined by immunofluorescencemicroscopy. As expected, the mLAMP/DCLR chimera lacking the lysosomaltargeting signal (stained with anti-mouse LAMP) was found at the plasmamembrane, in contrast to the lysosomal localization of the endogenoushuman LAMP (stained with anti-human LAMP) (FIGS. 10A-F). However, andsurprisingly, the mLAMP/Gag/DCLR chimera, was located in intracellularvesicles, partially colocalized with the endogenous LAMP of the humancell.

Immune Responses of Mice Inoculated with the LAMP/Gag/DCLR DNA Chimera

BALB/c mice (n=8) are immunized intramuscularly with 50 μg DNA (prime)followed in 3 weeks with 10 μg DNA (boost). The antibody titer ofsamples taken 2 weeks after the boost injection showed a high titerresponse to the LAMP/Gag/DCLR construct versus the absence of detectableresponse to wild type Gag encoded by the Gag DNA construct (FIGS. 11A,B, C1-C3). CD4+ mediated responses measured by IL-4 mRNA synthesis andELISA assay of IFN-γ production were comparable to the high levelresponse to the standard LAMP/Gag construct. In contrast, the CD8⁺responses to LAMP/Gag/DCLR, assayed by Gag-specific tetramer binding andintracellular IFN-γ staining, were consistently less than those elicitedby LAMP/Gag. This difference of low CD8⁺ and high CD4⁺ and antibodyresponses is subject to further investigation. There possibly is a Th2bias in the immune response to this DCLR chimera vaccine construct.

Example 6 AAV Vector Constructs

The following AAV vector DNA vaccine constructs were synthesized:AAV/LAMP (negative control); AAV/Gag (wild type Gag); AAV/LAMP/Gag(LAMP/Gag chimera); AAV/hDC-LAMP/Gag (hDC-LAMP/Gag chimera);AAV/LAMP/Gag/DCLR (LAMP/Gag/DCLR chimera).

Immunization Protocol, Initial Experiments

Two initial immunization studies have been performed under conditions ofour conventional protocol: DNA prime, 50 μg 1M at day 1, followed by DNAboost, 10 μg 1M, or AAV boost, 5×10⁹ genomic copies, at day 21. Animalswere also immunized only once with the AAV vector. Animals weresacrificed at day 31 for CD8⁺ response assays, at day 39-40 for CD4⁺response assays, and at day 90 for CD4⁺ and CD8⁺ assays. Bleedings weretaken before immunization and at 3-4 week intervals throughout theexperiment for antibody assays, and were continued at monthly intervalsfor 6 months to date with the remaining mice. Two independent mouseimmunization experiments have been initiated to date; one is at 6 monthsduration and the other at 2 months.

Immune response of Mice & Duration of Immunity, Initial Experiments

Cellular Immune Responses:

In general, under conditions of this protocol and with the assay timepoints as above, comparing naked DNA and the AAV vector as the boostimmunization, there was no clear advantage of the AAV boost as comparedto the naked DNA boost, up to day 90.

Antibody Responses

In keeping with the literature on the AAV vector system, it appears thatone obvious difference to naked DNA is a sustained humoral response,presumably due to long-term expression of the antigen (Table 1). Asobserved in our previous experiments, inoculation with the LAMP/gagchimera elicited a rapid Gag specific IgG response, with a titer in thisexperiment of 72,000 two weeks after the boost immunization. Withoutadditional immunizations this IgG response rapidly diminished to virtualbackground levels 3-4 months later. In contrast, immunization with theAAV vector resulted in a slower onset, but a sustained and much highertiter IgG response. Notably, after DNA prime, the AAV/LAMP/Gag/DCLRchimera, with a single AAV boost, resulted in a sustained IgG titer ofover 200,000, approximately 10-fold greater than the response to theAAV/LAMP/Gag construct. These data further suggest a Th2 bias of theDCLR system.

TABLE 1 HIV-1 Antibody Titers From Pooled Serum Of Immunized Mice*[Table did not reproduce] Day Vaccine 11 34 68 91 133 AAV WT — — — — —LAMP/Gag (DNA/DNA) — 72,000 900 300 — LAMP/Gag (DNA/AAV) — 2,700 8,10024,000 24,000 LAMP/Gag (AAV) — 8,100 2,700 2,700 2,700LAMP/Gag/DCLR (DNA/AAV) — 24,000 >218,000 >218,000 218,000ILAMP/Gag/DCLR (AAV) — 12,700 8,100 8,100 124,000 *The inoculationprotocol is indicated in the parenthesis, i.e., (DNA/DNA) indicates 2

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill in the artwithout departing from the spirit and scope of the invention and theclaims.

All of the patents, patent applications, international applications, andreferences identified are expressly incorporated herein by reference intheir entireties.

What is claimed is:
 1. A DNA vaccine comprising a chimeric vectorencoding a fusion protein which comprises an antigen domain comprisingat least one epitope and a trafficking domain and a pharmaceuticalcarrier.
 2. The DNA vaccine of claim 1, wherein the antigen domain isfused in frame with the trafficking domain.
 3. The DNA vaccine of claim1, wherein the trafficking domain directs both membrane and non-membraneproteins to an endosomal or lysosomal compartment in a cell or to alysosome-related organelle.
 4. The DNA vaccine of claim 1, wherein thetrafficking domain comprises a polypeptide trafficking domain of anendocytic receptor.
 5. The DNA vaccine of claim 1, wherein thetrafficking domain is the luminal domain of a lysosomal membranepolypeptide.
 6. The DNA vaccine of claim 5, wherein the lysosomalmembrane polypeptide comprises lysosome associated membrane protein(LAMP)-1, LAMP-2, LAMP-3, DC-LAMP, or LIMP polypeptide.
 7. A DNA vaccinecomprising a chimeric vector encoding a fusion protein which comprises aGag polypeptide fused in frame with a mammalian cellular polypeptide anda pharmaceutical carrier.
 8. A method for modulating an immune responsein an animal comprising administering the DNA vaccine of claim
 7. 9. Amethod for generating an immune response in an animal to an antigen,comprising: administering to the animal a cell comprising a vectorcomprising a nucleic acid molecule encoding a chimeric proteincomprising an antigen domain comprising at least one epitope, and atrafficking domain, wherein the cell expresses the chimeric protein inthe animal.
 10. The method according to claim 9, wherein the cellcomprises an MHC class II molecule compatible with MHC proteins of theanimal, such that the animal does not generate an immune responseagainst the MHC class II molecule.
 11. The method according to claim 9,wherein the animal is a human.
 12. A method for eliciting an immuneresponse to an antigen, comprising administering to an animal a cellcomprising a vector comprising a nucleic acid molecule encoding achimeric protein comprising an antigen domain comprising at least oneepitope, and a trafficking domain.
 13. The method according to claim 12,wherein the vector is infectious for a cell in the animal.
 14. Themethod according to claim 9, wherein the antigen is selected from thegroup consisting of: a portion of an antigenic material from apathogenic organism, a portion of an antigenic material from acancer-specific polypeptide, and a portion of an antigenic material froma molecule associated with an abnormal physiological response.
 15. Themethod according to claim 14, wherein the pathogenic organism is avirus, microorganism, or parasite.
 16. The method according to claim 15,wherein the virus is an HIV virus.
 17. The method according to claim 14,wherein the abnormal physiological response is an autoimmune disease, anallergic reaction, cancer, or a congenital disease.
 18. The methodaccording to claim 9, wherein a cell is obtained from the patient andwherein the vector is introduced into the cell and the cell or progenyof the cell is reintroduced into the patient.
 19. The method accordingto claim 18, wherein the cell obtained from the patient is a stem cellcapable of differentiating into an antigen presenting cell.
 20. The cellaccording to claim 19, wherein the antigen presenting cell does notexpress any co-stimulatory signals and the antigen is an auto-antigen.21. The method according to claim 19, wherein the cell does not expressany co-stimulatory signals and the antigen is an autoantigen.